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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 1st seminar shrimp inbreeding Presentation Transcript

  • Development of Two Microsatellite Multiplex Systems for Black Tiger Shrimp Penaeus monodonand Its Application in Genetic Diversity Study for Two PopulationsIbnu Sahidhir
  • 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
  • Introduction Microsatellite multiplex system ? Problem of Application and solution3 @IBNU SAHIDHIR www.artaquaculture.blogspot.com
  • 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
  • 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
  • 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
  • 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
  • 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
  • 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
  • Methods in molecular cloning Cutting and joining DNA fragment Multiplying DNA fragment Selecting DNA fragment Making Primer10 @IBNU SAHIDHIR www.artaquaculture.blogspot.com
  • 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
  • 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
  • Test the primers13 @IBNU SAHIDHIR www.artaquaculture.blogspot.com
  • 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
  • DNA Extraction 12,000 g 10 minutesTissue Extracted Cell Add by extractionsample Chloroform buffer 15 @IBNU SAHIDHIR www.artaquaculture.blogspot.com
  • Microsatellite Isolation: 1st Hybridization Microsatelli Microsate te llite16 @IBNU SAHIDHIR www.artaquaculture.blogspot.com
  • Microsatellite Isolation: 2nd Hybridization and Designing New Primer DNA sample17 @IBNU SAHIDHIR www.artaquaculture.blogspot.com
  • 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
  • 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
  • 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
  • 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
  • 1. Initial screening ofpublicly-available microsatellite sequences based on shrimp DNA test (i) 22 @IBNU SAHIDHIR www.artaquaculture.blogspot.com
  • 1. Initial screeningof publicly-available microsatellite sequences (ii)23 @IBNU SAHIDHIR www.artaquaculture.blogspot.com
  • 2a. With and Without Probe Hybridization Methods for New Microsatellite Isolation24 @IBNU SAHIDHIR www.artaquaculture.blogspot.com
  • 2b. New microsatellite loci25 @IBNU SAHIDHIR www.artaquaculture.blogspot.com
  • 3. Multiplex systems26 @IBNU SAHIDHIR www.artaquaculture.blogspot.com
  • 4a. Trial in Australian P. monodon Populations27 @IBNU SAHIDHIR www.artaquaculture.blogspot.com
  • 4b. Trial in Thailand P. monodon Populations28 @IBNU SAHIDHIR www.artaquaculture.blogspot.com
  • 4c. Trial in two P. monodon populations29 @IBNU SAHIDHIR www.artaquaculture.blogspot.com
  • 4d. P monodon’s Genetic Diversity Australia vs Thailand Australia Thailand30 @IBNU SAHIDHIR www.artaquaculture.blogspot.com
  • 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
  • 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 @IBNU SAHIDHIR www.artaquaculture.blogspot.com