Seed purity test


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Seed purity test

  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 - Purity test of Rice parents and hybrids using SSR markers
  3. 3. Genomics and Proteomics lab - Microsatellite markers polymorphism between parental lines and rice hybrids Tamilkumar et al.,2009
  4. 4. 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
  5. 5. Genomics and Proteomics lab - Amplification pattern of the parental lines obtained using the SSR marker RM202
  6. 6. 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).
  7. 7. 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
  8. 8. 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.
  9. 9. 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)
  10. 10. 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
  11. 11. 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)
  12. 12. 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.
  13. 13. Genomics and Proteomics lab - Thanks to • Dr R.Umarani • Dr Jerlin • Mr Tamil Kumar • Ms Padma Seed centre , TNAU