Molecular markers
PCR based
Requiring sequence
knowledge
courtesy of Carol Ritland
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PCR markers prior sequence
knowledge
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Microsatellites (SSR/STR/ STMS)
SSCP
ISSR
T-RFLP
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Microsatellite (SSR/STR/STMS)
• Also known as Short Sequence Repeat/Simple Tandem
Repeats/Sequence-Tagged Microsatellite Sites
• Repeats are 1 to 10 nucleotides bp long
• Mutation rate is higher than base rate (1X104 vs 1X108)
• Related to VNTR (minisatellites)
• PCR based
• Require extensive labour prior to finding useable
markers
• Can be expensive to find these markers
• Co-dominant
• Litt, M. and Lutty, J. A. Am. J. Hum. Genet. 1989
44:397-401
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Allelic Variation at a
Microsatellite Locus
GCCATGACACACACAGTAACGT
Allele “A”
Allele “B”
GCCATGACACACACACACACACACAGTAACGT
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Mechanisms of mutation
(slipped-strand mispairing)
Step 1
ca ca ca ca
gt gt gt gt gt gt gt
Step 2
Step 3
ca ca ca ca
gt gt gt gt gt gt gt
Step 4
ca ca ca ca ca ca
gt gt gt gt gt gt gt
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Development of SSR
•Construction of DNA library
•Restriction Enzyme digestion
•Ligation to plasmid
•Screening for various repeats (Southern blot)
•Sequencing positive clones
•Primers design to flank microsatellites
•Testing Primers for polymorphism (need
segregating families preferably with known
parents)
•Focal vs Non focal species
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Step by Step….
Restriction Digestion
Gel
electrophoresis
Alu I AGCT
Hae III GGCC
Rsa I GTAC
Isolate fragments
Ligase
Vector for cloning
200 to 500 bp
Cloning and screen for positive clones
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Step by Step cont’d
Screen for repeats
CA or CAT or CATA
CA positive clone
Using CACACA(25)
probe
1X106 clones
Primer design
from clones that
show repeats
Sequence all positive
clones usually 96 at a time
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SSR primer design
•50% contain repeats < 10 bp
•20% contain repeats starting at one end of the sequence
•20% to 30% contain repeats that may be usable
•Watch for complex repeats eg. Compound =
GCGGCCATATAT(16)GCGATGATATAT(16)GCGAA
Irregular = GCGGCCATATATCCATATAT(16)CCATATGCG
Complex = GCGGCCATATATCCATAT(12)GCTGCT(10)GCG
•Ideally design primers 18 to 24 nucleotides
•Aim for PCR product sizes that are > 100bp to 400bp
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Primer testing
• Require ideally >3 populations for testing
• Ideally 6 individuals randomly sample per
population
• 20% yield zero or poor amplicons
• 24% yield multiple or uninterruptible bands
• 18% monomorphic bands
• 38% usable microsatellite marker
• Squirrell et al. 2003 Mol. Ecol. 12:1339-1348
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SSR gel

= female parental type
WRC paternity analysis
= male parental type
A. Miscampbell
= size ladder
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Issues with Microsatellites (SSR)
•Highly variable and somatically stable marker
•Specific primers designed for target species (18-25 nt)
•Highly reproducible and yet evolve quickly (mutation rate
is higher than normal rates)
•A co-dominant marker with high heritability
•Excellent for paternity/pedigree analysis
•Could be difficult to use between species (focal vs non
focal species)
•Null alleles (lacking one of the allelic band for some
heterozyote individuals within a population) test with
family; excessive homozygotes, under estimate of
diversity
•Stutter bands (due to Taq incomplete amplification)
•Subjectiveness when scoring (be consistent)
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Scoring microsatellites
• Require known ladder to run with samples
• Resolve 2 or more bases differences using
polyacrylamide gel
• Use base size to score allelic differences
Sample
Cat A
Cat B
Cat C
Locus A
202
200
202
Locus A’
204
206
206
Locus B
353
353
351
Locus B’
355
357
355
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Score SSR gel:
Samples = 21
204 bp
200 bp
175 bp
145 bp
120 bp
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2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
A1
A2
B1
B2
C!
C2
D1
D2
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Allelic variation and statistical analyses
•Matala, A.P., Gray, A.K.,
Heifetz, J. and Gharrett,
A.J. (2004) Envior. Biololy
of Fishes 69:201-210
•Population structure of
Shortkaker Rockfish
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Example of allelic variation in
microsatellites
Microsatellite
variation and
genetic
relationship
among
Rajasthani
sheep:
Relevance for
conservation
R Arora and S
Bhatia
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