1st seminar shrimp inbreeding

Development of Two Microsatellite Multiplex Systems
for Black Tiger Shrimp Penaeus monodon
and Its Application in Genetic Diversity Study for Two
Populations
Ibnu Sahidhir

Content
Introduction
 Microsatellite multiplex system ?
 Function, Problem and solution
Material and Methods
 Test the available
 Isolate new microsatellite markers
 Develop two microsatellite multiplex sytem
 Asses genetic diversity
Results and Discussion
Conclusion
Personal Comment

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www.artaquaculture.blogspot.com

Introduction
Microsatellite multiplex system ?
Problem of Application and solution

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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)

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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 alltogether

Plus: reduces the time and costs
Minus: More complex to analyse the result ?

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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

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)

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Problems and solutions

Present 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. Monodon

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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
shrimp

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Methods in molecular cloning

Cutting and joining
DNA fragment

Multiplying DNA fragment

Selecting DNA fragment
Making Primer
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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

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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.
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Test the primers

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Isolate new microsatellite markers
1. Extract the DNA
2. Cut and join the DNA (restriction and ligation)
3. Amplify the DNA fragment by PCR
4. Hybridize the amplicons to probe filter (2 probe, 2 temperature)
5. Recombine and clone the DNA (TOPO TA cloning)
6. Reculture the clones
7. Lyse the clones
8. Hibridize with fluerescens probes
9. Sequence the positive clones
10. Make primers
11. Test in sample DNA

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DNA Extraction

12,000 g 10
minutes

Tissue Extracted Cell
Add
by extraction
sample Chloroform
buffer

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Microsatellite Isolation: 1st Hybridization
Microsatelli
Microsate te
llite

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Microsatellite Isolation:
2nd Hybridization and Designing New Primer
DNA
sample

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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

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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

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Results and Dicussion
Summary of Result
Choose and test suitable
microsatellites:
Develop new
microsatellites:
Combine suitable
microsatellites primer to new suitable
make 2 microsatellites microsatellites
multiplex system

Test to Australian and Thailand
shrimp

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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:
Combine 13 microsatellites
primer to make 2 7 new suitable
microsatellites multiplex microsatellites
system

Australian shrimp genetic
diversity better than Thailand’s
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1. Initial screening of
publicly-available microsatellite sequences based on shrimp
DNA test (i)

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1. Initial screening
of publicly-available microsatellite sequences (ii)

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2a. With and Without Probe Hybridization
Methods
for New Microsatellite Isolation

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2b. New microsatellite loci

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3. Multiplex systems

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4a. Trial in Australian P. monodon Populations

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4b. Trial in Thailand P. monodon Populations

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4c. Trial in two P. monodon populations

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4d. P monodon’s Genetic Diversity
Australia vs Thailand

Australia

Thailand

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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

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)

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