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DNA Markers Techniques for Plant Varietal Identification

How to use various kinds of markers for seed purity test is explained in this presentation

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DNA Markers Techniques for Plant Varietal Identification

  1. 1. Genomics and Proteomics lab - DNA Markers Techniques for Plant Varietal IdentificationDr.N.SenthilAssociate Professor ( Biotechnology)Genomics and proteomics lab N. Senthil, 2P.Tamilkumar , 3M. Raveendran, 4R. Jerlin and 5R. Umarani &3 Centres for Plant Molecular Biology, TNAU, Coimbatore-3 2 Department of Seed Science and Technology, TNAU, Coimbatore-3. 4&5 .Seed Centre, Tamil Nadu Agricultural University, Coimbatore-3
  2. 2. Genomics and Proteomics lab - Genomics time line
  3. 3. Genomics and Proteomics lab - www.tnaugenomics.comComparing genomes: Example from the grasses This is now one of the most well-known figures in plant comparative genomics. This consensus comparative map of 7 grasses shows how the genomes can be aligned in terms of “rice linkage blocks” (Gale and Devos 1998). Any radial line starting at rice, the smallest genome and innermost circle, will pass through regions of similar gene content in each of the other species. Therefore a gene on the chromosome of one grass species can be anticipated to be present in a predicted location on a specific chromosome of a number of other grass family species. This has facilitated much sharing among researchers working on any of these species and others that may be also related (Phillips & Freeling 1998).
  4. 4. Genomics and Proteomics lab - Automated sequencing reactions - each reaction can resolve 600 to 750 bp (labeled with fluorescent dyes)
  5. 5. Genomics and Proteomics lab - SNP discovery- Early methods • Re-sequencing of PCR amplicons with or without pre-screening • Direct sequencing of DNA segments amplified by PCR)from several individuals is the most direct way to identify SNP polymorphisms • Alternatively, an allele-specific-PCR or primer-extension assay may be developed relatively straightforwardly. Rafalski 2002 Curr Opin Plant Biol 5 :94-100
  6. 6. Genomics and Proteomics lab - DNA sequencing output If you have DNA sequence produced from a PCR product or a library of ESTs, the sequence of your DNA segment(s) will be given to or, more usually, emailed or electronically transferred to you.. If the data is in the chromatogram form, you will need to manually generate a text file such as the one below (by “reading” the bases yourself) or, more typically, use one of the many software programs available to do this for you. If you retrieve a sequence from a public database, it will already be in this format for you. The first 480 bases of the DNA sequence of GAN, a drought tolerance related gene in Arabidopsis (GenBank Accession AY986818).
  7. 7. Background information : Markers• The identification of varieties of agricultural crops is important at every stage of the seed production chain (Cooke, 1995).• The only legally recognized method in our country for genetic purity assessment based on field plot grow out tests, which include only the morphological traits• But morphological traits may not be sufficient for discrimination of all new varieties and hybrids, they also subjected to environmental influences and requires one full season .
  8. 8. Genomics and Proteomics lab - What are molecular markers? • A marker, in this context, is an identifier (sometimes called a “tag”) of a particular aspect of phenotype and/or genotype; its inheritance can easily be followed from generation to generation. • Markers can be: Morphological: phenotypic variation which is scorable on the basis of a single plants (e.g. flowering time) • Biochemical: variants in the size or net charge of a protein (eg isozymes) or in the chemical composition of a metabolite (e.g. sugar) • Molecular: variants in the DNA sequence (eg microsatellites)
  9. 9. Genomics and Proteomics lab - Molecular markers : Costs • Using molecular markers requires the use of specific laboratory equipment, at the very least a PCR (polymerase chain reaction) thermalcycler and electrophoresis and visualization equipment. • So start-up costs can be high, although these may be compensated for by later savings (and prices of the necessary equipment and reagents have been decreasing over time).
  10. 10. Genomics and Proteomics lab - Molecular markers : Technical skills needed • Along with the equipment required for molecular marker work comes the need for the technical skills and knowledge of how to do the work and understand the results. • These are not difficult skills to learn, but are not always part of a classical plant breeder’s education.
  11. 11. Genomics and Proteomics lab - Considerations in selection of marker type • There are many types of molecular markers available. Which type you select to use for your project will depend on: • What the goals of the project are • How variable the germplasm is • What sort of population is being analyzed • What level of resolution is needed • Whether or not there is previous work you can take advantage of (ie. Marker development)
  12. 12. Genomics and Proteomics lab - Classification of DNA markers Probe Based (eg. RFLP)  Restriction fragment length polymorphism (RFLP) Amplification Based (eg. RAPD, SSR,ISSR,SCAR)  Random amplified polymorphic DNA (RAPD)  Simple sequence repeats (SSR)  Inter Simple Sequence Repeats (ISSR)  Sequenced Characterized Amplified Region Markers (SCAR) Combination of probe based and PCR based markers (eg. AFLP)  Amplified fragment length polymorphism (AFLP) New Generation markers  Single Nucleotide Polymorphism (SNP)
  13. 13. Genomics and Proteomics lab - Relative importance of molecular markers (Christian Schlotterer. 2004 )
  14. 14. Genomics and Proteomics lab - RFLP(Restriction Fragment Length Polymorphism)
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  17. 17. Genomics and Proteomics lab - PCR Schematic drawing of the different steps of polymerase chain reaction (PCR): (a) denaturing step at 92-95°C; (b) primer annealing step (37-68°C depending of the technique); (c) extension step at 72°C (P=Taq DNA polymerase), and (d) end of the first cycle with two copies of DNA strands. The two resulting DNA strands make up the template DNA for the next cycle, thus doubling the amount of DNA duplicated for each new cycle.
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  19. 19. Genomics and Proteomics lab - AFLP(Amplified Fragment Length Polymorphisms) Developed in the early 1990’s by Keygene Combination of both RFLP and PCR techniques Four steps  DNA is digested with two different restrictionenzymes  Oligonucleotide adapters are ligated to the endsof the DNA fragments  Specific subsets of DNA digestion products are amplified, using combinations of selective primers  Polymorphism detection is possible with radioisotopes, fluorescent dyes or silver staining
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  22. 22. Genomics and Proteomics lab - Genome organization • It is important to remember that only part (sometimes a very small part!) of the DNA sequence is composed of genes. • The rest is non-coding sequence, including lots of repetitive sequences, microsatellites and transposons. In some species, the genic fraction of the genome may be <10% of the total.
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  24. 24. Genomics and Proteomics lab - SSR Single Sequence Repeat
  25. 25. Genomics and Proteomics lab - flanking region I flanking region II microsatellite plant A plant B specific primers were designed corresponding to flanking sequence of microsatellite plant A plant B PCR analysis and analyze on 6 %denaturing polyacrylamide gel with silver stainingSchematic of SSR A B Repetitive sequence primer Iassay primer II
  26. 26. Genomics and Proteomics lab - SSR •
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  28. 28. Genomics and Proteomics lab - Inter-Simple Sequence Repeat (ISSR) • Relies on one primer for PCR that anneals to an SSR region and amplifies region between inversely oriented adjacent SSRs • Can be undertaken for any species that contains a sufficient number and distribution of SSR motifs • Genomic sequence data not required • Amplifies large numbers of DNA fragments per reaction representing multiple loci from across the genome • Ideal method for fingerprinting varieties
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  30. 30. Genomics and Proteomics lab - ISSR
  31. 31. Genomics and Proteomics lab - Genetic Diversity of Indian Jatropha Species
  32. 32. Genomics and Proteomics lab - SCAR Marker
  33. 33. Genomics and Proteomics lab - Sequence Characterized Amplified Regions (SCAR)
  34. 34. Genomics and Proteomics lab - Cleaved Amplified Polymorphic Sequence (CAPS) • Primers used to amplify the genome segment of interest • Amplification product digested with selected restriction enzyme • Digest is subjected to agarose gel electrophoresis to detect polymorphism in the lengths of fragments generated • Codominant marker • Quick assay
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  39. 39. Genomics and Proteomics lab - Purity test of Rice parents and hybrids using SSR markers
  40. 40. Genomics and Proteomics lab - Microsatellite markers polymorphism between parental lines and rice hybrids Tamilkumar et al.,2009
  41. 41. Genomics and Proteomics lab - Microsatellite markers polymorphism between parental lines and rice hybrids • Five microsatellite markers RM276, RM 234, RM 258, RM202 and RM 204 together differentiated 5 hybrids and the parental lines at least with a single marker allele difference. • The microsatellite marker, RM234 amplified alleles specific to differentiate parental lines of CORH3 likewise RM276 for KRH2, RM258 for PRH10, RM202 for AJAY and RM204 for RAJALAXMI used to differentiate parental lines of respective hybrids. Tamilkumar et al.,2009
  42. 42. Genomics and Proteomics lab - Amplification pattern of the parental lines obtained using the SSR marker RM202
  43. 43. Genomics and Proteomics lab - Testing genetic purity of hybrid seeds of CORH3 using the SSR marker RM 234 • Genomic DNA was isolated from 50 seedlings of the CORH3 hybrid (random sample) • PCR analysis was performed by means of the RM234 out of 50 random samples microsatellite marker identified presents of single pollen shedder (B line) seed, which had a CMS line specific fragment • This amounts to 2% off types in the hybrid seed produced . • The results were confirmed using 400 seeds from the same seed lot through Grow out test (GOT).
  44. 44. Genomics and Proteomics lab - Testing genetic purity of hybrid seeds of CORH3 using the SSR marker RM 234 Lane 2 = TNAUCMS2A (CMS line), Lane 3 = CB87R (restorer line). DNA was isolated from single seedlings of the CORH3 hybrid, PCR analysis was performed and genotype assessed (Lanes 4–12) Off type in Lanes 8. Tamilkumar et al.,2009
  45. 45. Genomics and Proteomics lab - www.tnaugenomics.comSeed Purity Assessment Of Rice Hybrid UsingMicrosatellite Markers Arrow indicates contaminants Yashitola et al.,2002 Detection of impurities in the Indian rice hybrid-KRH2 Through multiplex PCR using the microsatellite markers RM164 and RM206. M—50 bp ladder, A—CMS line (IR58025A), H— Hybrid (KRH2), R—Restorer line (KMR3), 221 to 240— Samples of hybrid KRH2 collected from a commercial seed-lot.
  46. 46. Genomics and Proteomics lab - SSR (Multiplex) Multiplex PCR assay for distinguishing rice hybrids using three SSR markers Lane C1-IR58025A, lane R1-IR40750R, lane H1-DRRH1, lane C2- IR58025A, lane R2-KMR3R, lane H2-KRH2, lane C3-IR58025A, lane R3-C20R, lane H3-CORH2, lane C4- IR58025A, lane R4-BR827-35R, lane H4-Sahyadri (Sundaram et al., 2007)
  47. 47. Genomics and Proteomics lab - www.tnaugenomics.comSSR markers utilization in seed purity assessment ofIR58025A Sundaram et al., 2007 Two-dimension assay involving a 20 *20 grow-out matrix for assessment of purity of IR58025A with the help of SSR markers RM202 and RM276. (a) Row-wise lanes 6 & 8 and Column-wise lanes 3 & 18 (indicated by arrows) represent contaminants. (b) Schematic representation of the 20 *20 matrix based method for rapid identification of contaminants in IR58025A. Plants at intersections of 6th row 18th column and 8th row and 3rd column (indicated by arrow) were identified as contaminants
  48. 48. Genomics and Proteomics lab - www.tnaugenomics.comComparison of the most used marker systems Feature RFLPs RAPDs AFLPs SSRs SNPs DNA required 10 0.02 0.5-1.0 0.05 0.05 (μg) DNA quality High High Moderate Moderate High PCR based No Yes Yes Yes Yes Number of 1.0-3.0 1.5-50 20-100 1.0-3.0 1.0 polymorph Loci analysed Ease of use Not easy Easy Easy Easy Easy Amenable to Low Moderat Moderate High High automation e Reproducibilit High Unreliab High High High y le Development Low Low Moderate High High cost Cost per High Low Moderate Low Low analysis (Korzun et al.,2001)
  49. 49. Genomics and Proteomics lab - Conclusion • DNA profiling could be used now for the verification or confirmation of varietal identity and in some quality control situations. • DNA profiling methods for statutory variety registration is still under discussion between UPOV and other interested parties.
  50. 50. Genomics and Proteomics lab - Thanks to • Dr R.Umarani • Dr Jerlin • Mr Tamil Kumar • Ms Padma Seed centre , TNAU