1st seminar shrimp inbreeding


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  • 1 bp difficult to ampifly5 and 6 bp in few proportion
  • Different environment may be make different flanking region therefore need different primer
  • 10 mM Tris–HCl pH 8 = buffer pH, 100 mM EDTA=inhibit DNAase, 10 mM NaCl =reduce ionic interaction DNA-kation, 1% SDS (sodium dodecyl sulfate)=separate DNA-protein, and 250 μ g/ml Proteinase K=disrupt cell membranes. Sodium chloride=reduce ionic interaction, beta-mercaptoethanol=reduce foam and stabilize interface. Chloroform=denature protein, DNA-protein
  • PCR cycling protocol: 95 °C for 15 min, 30 cycles of 94 °C for 30 s, 60 °C for 90 s and 72 °C for 60 s, a final extension at 60 °C for 30 min
  • Exclusion of dinucleotide repeatAvailability of flanking regionsEstimated product size range (100-350 bp)
  • 1st seminar shrimp inbreeding

    1. 1. Development of Two Microsatellite Multiplex Systems for Black Tiger Shrimp Penaeus monodonand Its Application in Genetic Diversity Study for Two PopulationsIbnu Sahidhir
    2. 2. ContentIntroduction Microsatellite multiplex system ? Function, Problem and solutionMaterial and Methods Test the available Isolate new microsatellite markers Develop two microsatellite multiplex sytem Asses genetic diversityResults and DiscussionConclusionPersonal Comment 2 @IBNU SAHIDHIR www.artaquaculture.blogspot.com
    3. 3. Introduction Microsatellite multiplex system ? Problem of Application and solution3 @IBNU SAHIDHIR www.artaquaculture.blogspot.com
    4. 4. What is Microsatellite ? Multiple copies of simple sequence: size 1- 6 bp, <few hundred repeat (AC)15  (ATA)8  (CACG)7 = AC…AC15 = = CACG…CACG7 (CA)8(CG)12 ATA…ATA 8 Flanking Primer target Microsatellite region (redish (bold) (AG…) color) 4 @IBNU SAHIDHIR www.artaquaculture.blogspot.com
    5. 5. Microsatellite multiplex system?• Co-amplification of two or more loci in a single PCR reaction• Using >1 primer to annealing microsatellite in amplification process• Efficient, but need trial to make primers work alltogetherPlus: reduces the time and costsMinus: More complex to analyse the result ? 5 @IBNU SAHIDHIR www.artaquaculture.blogspot.com
    6. 6. What is its benefits ? Prevent overestimates of genetic variation ex: Nemoria arizonaria (catkins in spring and twig in summer) (phenotypic plasticity: different phenotype expressed by the same gene(s)). Very useful for selective breeding program (prevent inbreeding, genetic diversity reduction, parental tracking) 6 Making easy to calculate quantitative SAHIDHIR (QTL) @IBNU trait loci www.artaquaculture.blogspot.com
    7. 7. How does it works ? (Hayes and Andersen, 2005) Highly polymorphic (Individuals within species differ in number repeat of simple sequences in same locus, e.g. (AC)10 and (AC)15) Heritable Codominant (could be distinguished between homozigotes and heterozigotes) 7 @IBNU SAHIDHIR www.artaquaculture.blogspot.com
    8. 8. Problems and solutionsPresent Condition of Microsatellite Application in P. Monodon Culture Application single-locus microsatellite is less proper for asses genetic diversity in wider geographic area Unassesed published available microsatellite for multiplex system Aims:  Analyse publicly available microsatellite of P. Monodon  Isolate new microsatellite markers  Develop two microsatellite multiplex sytem  Apply it to asses genetic diversity of two population of P. Monodon8 @IBNU SAHIDHIR www.artaquaculture.blogspot.com
    9. 9. Materials and Methods Principle methods in molecular cloning Choose and test suitable microsatellites: Develop new microsatellites: 2 step enrichment library Combine suitable microsatellites primer to new suitable make 2 microsatellites microsatellites multiplex system Test to Australian and Thailand shrimp9 @IBNU SAHIDHIR www.artaquaculture.blogspot.com
    10. 10. Methods in molecular cloning Cutting and joining DNA fragment Multiplying DNA fragment Selecting DNA fragment Making Primer10 @IBNU SAHIDHIR www.artaquaculture.blogspot.com
    11. 11. Test of published microsatellite Analysis publicly available microsatellite of P Monodon Repeats (types, number) Flanking sequences Size product (bp) Making primer Tested to sample shrimp DNA Numbers of alleles Reproducibility of result Ease for scoring data 11 @IBNU SAHIDHIR www.artaquaculture.blogspot.com
    12. 12. Designing Primer Assessed the presence of tri or tetranucleotide repeats, suitability of flanking sequence for primer design, from 90 sequences containing microsatellites of monodon GenBank database. Designed using PRIMER v.3, based upon the guidelines for multiplex primer design (Multiplex polymerase chain react on (PCR) Handbook 09/2002, Qiagen. 12 @IBNU SAHIDHIR www.artaquaculture.blogspot.com
    13. 13. Test the primers13 @IBNU SAHIDHIR www.artaquaculture.blogspot.com
    14. 14. Isolate new microsatellite markers1. Extract the DNA2. Cut and join the DNA (restriction and ligation)3. Amplify the DNA fragment by PCR4. Hybridize the amplicons to probe filter (2 probe, 2 temperature)5. Recombine and clone the DNA (TOPO TA cloning)6. Reculture the clones7. Lyse the clones8. Hibridize with fluerescens probes9. Sequence the positive clones10. Make primers11. Test in sample DNA 14 @IBNU SAHIDHIR www.artaquaculture.blogspot.com
    15. 15. DNA Extraction 12,000 g 10 minutesTissue Extracted Cell Add by extractionsample Chloroform buffer 15 @IBNU SAHIDHIR www.artaquaculture.blogspot.com
    16. 16. Microsatellite Isolation: 1st Hybridization Microsatelli Microsate te llite16 @IBNU SAHIDHIR www.artaquaculture.blogspot.com
    17. 17. Microsatellite Isolation: 2nd Hybridization and Designing New Primer DNA sample17 @IBNU SAHIDHIR www.artaquaculture.blogspot.com
    18. 18. Develop two microsatellite multiplex sytem Label the new primers with fluorescent dyes Combine the primers Scorability Size product (bp) Asses it with PCR Analyse the result 18 @IBNU SAHIDHIR www.artaquaculture.blogspot.com
    19. 19. Testing New Microsatellite Multiplex System to Australia and Thailand Black Tiger Shrimp Population Sampling the shrimp DNA of both population Checking it with multiplex PCR Analysing the genetic diversity Analysing heterozygosity and extent of genetic differentiation with GENEPOP 3.4. Discriminating the subgrpoup with NTSYSpc Analysing the genetic distance by Lynch coefficient 19 @IBNU SAHIDHIR www.artaquaculture.blogspot.com
    20. 20. Results and Dicussion Summary of Result Choose and test suitable microsatellites: Develop new microsatellites: 2 step enrichment library Combine suitable microsatellites primer to new suitable make 2 microsatellites microsatellites multiplex system Test to Australian and Thailand shrimp20 @IBNU SAHIDHIR www.artaquaculture.blogspot.com
    21. 21. Summary 90 publicly-available microsatellites Exclusion of dinucleotide repeat Availability of flanking regions Estimated product size range (100-350 bp) 19 chosen  Assesed by 15 Australian P. Monodon  7, did not amplify: different priming sites 6 suitable  6, wide range of allele size microsatellites: Small number ! Develop new microsatellites: 2 step enrichment libraryCombine 13 microsatellites primer to make 2 7 new suitable microsatellites multiplex microsatellites system Australian shrimp geneticdiversity better than Thailand’s 21 @IBNU SAHIDHIR www.artaquaculture.blogspot.com
    22. 22. 1. Initial screening ofpublicly-available microsatellite sequences based on shrimp DNA test (i) 22 @IBNU SAHIDHIR www.artaquaculture.blogspot.com
    23. 23. 1. Initial screeningof publicly-available microsatellite sequences (ii)23 @IBNU SAHIDHIR www.artaquaculture.blogspot.com
    24. 24. 2a. With and Without Probe Hybridization Methods for New Microsatellite Isolation24 @IBNU SAHIDHIR www.artaquaculture.blogspot.com
    25. 25. 2b. New microsatellite loci25 @IBNU SAHIDHIR www.artaquaculture.blogspot.com
    26. 26. 3. Multiplex systems26 @IBNU SAHIDHIR www.artaquaculture.blogspot.com
    27. 27. 4a. Trial in Australian P. monodon Populations27 @IBNU SAHIDHIR www.artaquaculture.blogspot.com
    28. 28. 4b. Trial in Thailand P. monodon Populations28 @IBNU SAHIDHIR www.artaquaculture.blogspot.com
    29. 29. 4c. Trial in two P. monodon populations29 @IBNU SAHIDHIR www.artaquaculture.blogspot.com
    30. 30. 4d. P monodon’s Genetic Diversity Australia vs Thailand Australia Thailand30 @IBNU SAHIDHIR www.artaquaculture.blogspot.com
    31. 31. Conclusion Publicly available microsatellite must be tested before application for species in different region. Two step hybridization improve succes of new microsatellite isolation. Microsatellite multiplex sytem could asses genetic diversity in two different region. Wild-type Australian black tiger shrimp has better genetic diversity than Thailand 31 @IBNU SAHIDHIR www.artaquaculture.blogspot.com
    32. 32. Personal comment Sampling more than one pond (in single pond, shrimps relatively come from identical genetic background) Comparing wild type vs wild type shrimp broodstock (not with cultured broodstock) 32 @IBNU SAHIDHIR www.artaquaculture.blogspot.com
    33. 33. 33 @IBNU SAHIDHIR www.artaquaculture.blogspot.com