1. PCR based Requiring sequence knowledge
Molecular markers
PCR based
Requiring sequence
knowledge
courtesy of Carol Ritland

2. PCR markers prior sequence knowledge
Microsatellites (SSR/STR/ STMS)
SSCP
ISSR
T-RFLP

3. 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 :

4. Allelic Variation at a Microsatellite Locus
GCCATGACACACACAGTAACGT
Allele “A”
Allele “B”
GCCATGACACACACACACACACACAGTAACGT

5. Mechanisms of mutation (slipped-strand mispairing)
Step 1
Step 2
Step 3
Step 4
ca ca ca ca
gt gt gt gt gt gt gt
ca ca ca ca ca ca

6. 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

7. Step by Step…. Restriction Digestion Gel electrophoresis Alu I AGCT
Hae III GGCC
Rsa I GTAC
Isolate fragments
Vector for cloning
Ligase
200 to 500 bp
Cloning and screen for positive clones

8. Step by Step cont’d CA positive clone Screen for repeats
Using CACACA(25)
probe
Screen for repeats
CA or CAT or CATA
1X106 clones
Primer design from clones that show repeats
Sequence all positive
clones usually 96 at a time

9. 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

10. 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 Mol. Ecol. 12:

11. SSR gel  = female parental type = male parental type = size ladder
WRC paternity analysis A. Miscampbell

12. 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)

13. 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
Locus A
Locus A’
Locus B
Locus B’
Cat A
202
204
353
355
Cat B
200
206
357
Cat C
351

14. Score SSR gel: Samples = 21 204 bp 200 bp 175 bp 145 bp 120 bp 1 2 3 4
175 bp   
145 bp
120 bp 1 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

15. Allelic variation and statistical analyses
Matala, A.P., Gray, A.K., Heifetz, J. and Gharrett, A.J. (2004) Envior. Biololy of Fishes 69:
Population structure of Shortkaker Rockfish

16. Example of allelic variation in microsatellites
Microsatellite variation and genetic relationship among Rajasthani sheep: Relevance for conservation
R Arora and S Bhatia