2. Contents
Problems of groupers brood stocks management
Objectives
Determine the way to solutions
Facts and explanations
Core messages and Implications
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4. “Whichever I choose, it will be the same.” “I’ll choose you based on my needs.”
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5. Three categories to measure genetic diversity
1. Physical performance
2. Biochemical indicator
3. DNA 1. Mitochondrial DNA
2. Nuclear DNA
In microsatellites relatively occurs more mutation
Means: more variations
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8. Male Female
1 1
2 2
2 x 2 = 4 pairs
4 broodstocks
1 x 3 = 3 pairs
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9. Nature Genetic makeup Selection
Good quality
Brood stock
1. Variation
Nurture
(Environment, nutrition)
2. Effective numbers of broodstock
3. Consider
Keep the variation
Unproductive broodstock
Microsatellites to measure
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10. “Microsatellites are repetitions of simple DNA sequences”
TTTTTTT AAAAAAAA AGAGAGAG
Primer target Flanking region Microsatellite
(bold part) (red part) (AG…)
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11. This short
DNA make
me sick !
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12. 2. Point out the genetic diversity
To answer: is it genetically variable ?
1. Develop the tools
To answer:
What kinds of 3. Evaluate communal spawning on
microsatellites primer
genetic diversity
will be effective ?
To answer: is unproductive broodstock
influence genetic diversity ?
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14. Evaluate in
population
Multiply them
Extract in PCR
microsatellites
Find their pattern using DNA sequencer
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18. a. Allelic diversity (A)
2. Point out
the genetic diversity b. Heterozygosity (H)
c. Polymorphic Information Content (PIC)
3. Evaluate communal
spawning on genetic d. Genetic relatedness (rxy)
diversity
e. Inbreeding Coefficient (FIS)
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20. a. Allelic diversity (A) a. Higher is better
b. Heterozygosity (H) b. Higher is better
c. Polymorphic Information Content (PIC) c. Higher is better
d. Genetic relatedness (rxy) d. Higher is better (absolute number
of expected and observed)
e. Inbreeding Coefficient (FIS) e. Negative is better
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21. “8 microsatellites are polymorphic”
1. The tools The tools power
Observed heterozygosity
Expected heterozygosity
Polymorphic information content
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22. 2. Coefficient of inbreeding (Fis = (He – Ho) / He)
Deviation between expected and observed
Positive (Hexpected-Hobserved) =
Fis > 0 = inbreeding is more than expected
Fis < 0 = inbreeding is low than expected 1. selection,
2. assortative mating,
3. migration,
4. null alleles
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23. 3. Genetic variation within population
Means allele per locus Means effective allele per locus
Allele richness
Expected heterozygosity Genetic relatedness
Observed heterozygosity Inbreeding coefficient
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24. 4. Genetic distance between stock
P (parents) G1 (offspring) PP (wild)
P (parents) 0.10781
G1 (offspring) 0.0781 0.1891
PP (wild)
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25. 5. Parentage analysis
P (Parents)
20 ind
13 Female x 7 male
= 91 pairs possibilities
G1 (offspring)
120 ind
105 ind (87.5%) full-sib = 1 pair
15 inds come from 4 pair
Proportion no female mating = (13-5) /13 pairs
>62% female have no chance to mate !
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26. 6. Ne = effective number of broodstock
F (inbreeding coefficient) =1/2Ne
P =30
G1 =13.8
PP =18
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27. 1. Microsatellites is able to show genetic diversity within and between population
2. a. Parents show higher genetic diversity, followed by new parents and the offspring
b. New parent brood stocks have lower genetic variation than the old one
3. Communal spawning highly reduce the effective population number of the offspring
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28. 1. Inbreeding in this paper is not necessarily means pedigree inbreeding (F) but
degree relatedness /homozygosity of genotype.
Namely Inbreeding as nonrandom mating and population subdivision inbreeding
(Keller and waller, 2002; Braude and Templeton, 2009)
2. Inbreeding is not necessary useless, purpose-based inbreeding for specific traits is
important (Tave, 1999).
3. Not only inbreeding depression, outbreeding depression sometimes is also happened
(Fenster and Galloway, 1999)
4. Need to take sample of the offspring for several cycle production
Population
(because 38% productivity of female is quite common)
Sub
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29. Variation
Wealth Number
Power Contribution
Woman
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32. GAC CACG GCAT CG CGA
GAC CACG substitution G CAT insertion CGCGA deletion
Before DNA replication
DNA replication process
After DNA replication
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33. Microsatellites has important functions
Regulate gene activity
Regulate metabolic process
Stabilize chromosomes structure
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34. A
T Microsatellites are located spreading in whole chromosomes
G
C
Microsatellites
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35. a. Collect sample Summary of methods
1. Develop the tools b. Extract DNA
c. Develop microsatellites primers
a. Allelic diversity (A)
2. Point out
the genetic diversity
b. Heterozygosity (H)
c. Polymorphic Information Content (PIC)
e. Genetic relatedness (rxy)
3. Evaluate communal
spawning on genetic d. Inbreeding Coefficient (FIS)
diversity
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38. Examples for excessive homozygosity
Expected heterozygosity Observed heterozigosity
A = 0.2 A = 0.6
B = 0.2 B = 0.1
C = 0.2 C = 0.1
D = 0.2 D = 0.1
E = 0.2 E = 0.1
=1 =1
He = 1- (0.04+0.04+0.04+0.04+0.04) Ho = 1- (0.36+0.01+0.01+0.01+0.01)
= 1-0.2 = 1 – 0.4
= 0.8 = 0.6
Fis = (0.8-0.6)/0.6
= 0.33
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39. Methods in molecular cloning
Cutting and joining DNA
fragment
Multiplying DNA fragment
Selecting DNA fragment
Making Primer
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40. 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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41. 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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42. Test the primers
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43. 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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44. DNA Extraction
12,000 g 10
minutes
Tissue Extracted Cell by Add
sample extraction buffer Chloroform
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45. Microsatellite Isolation: 1st Hybridization
Microsatelli
Microsatel te
lite
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47. 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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Editor's Notes
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
