1. A Genetic Analysis of the Local Rana sylvatica Population
The Wood Frog (Rana sylvatica)

2. Overall Goals
Determine relatedness of tadpoles within and between two ponds
Use these data to characterize
population structure
genetic health
behavior

3. How will we determine relatedness? Microsatellites
[CATA]n
unique flanking sequence
unique flanking sequence
repeat region
Di-, tri-, or tetra- nucleotide repeats that differ in # of repeats
A specific length is a specific “allele”
Genetically neutral
Flanking DNA is conserved throughout species

4. Determining microsatellite alleles in an individual
1) Polymerase chain reaction (PCR)
requires
-Taq DNA polymerase
-dNTPs
-primers (short, ssDNA)

5. Determining microsatellite alleles in an individual
1) Polymerase chain reaction (PCR)
2) Use (fluorescently-labeled) primers specific for flanking sequences
[CATA]n

6. Determining microsatellite lengths
3) Fragment analysis with automated sequencer
4) Corroborate with PAGE

7. Sample Fragment Analysis
black, MS
red, size standard

8. Hardy-Weinberg Principle
Assume a population with
random mating
no mutation
no migration
no natural selection

9. Hardy-Weinberg Principle
Assume a population with
random mating
no mutation
no migration
no natural selection
The allele and genotype frequencies of the population remain constant
= Hardy-Weinberg Equilibrium (HWE)

10. Questions (pp )
1) Do the frequencies of the microsatellite alleles in the Rana population significantly deviate from HWE?
Will be done for you and discussed Week 4

11. Questions (pp )
2) Is there evidence of inbreeding in either subpopulation?
Involves tabulating expected heterozygosity
Remember…
exp frequency heterozygotes
= (1 - exp frequency of homozygotes)

12. Questions (pp )
3) Is genetic drift a significant force driving divergence between the two subpopulations?
Genetic drift
= random divergence of allele frequencies
between subpopulations

13. Questions (pp )
3) Is genetic drift a significant force driving divergence between the two subpopulations?
Genetic drift
= random divergence of allele frequencies
between subpopulations
High in island model (low population/migration)
Low in panmictic model (high pop/mig)
Migration rate will be calculated in Question #4

14. Questions (pp. 91-96) 5) How many fathers per egg cluster?
Video: many males competing for a female
“pirate” males have been reported for another Rana species
(Hint: Next prelab)

15. Rana tadpole sibling interactions
aggregation
repulsion
Molecular Ecology (2006) 15,

16. Questions (pp )
6) How do tadpole siblings position themselves relative to each other?
Question 5 of next prelab asks you to design an experiment.

17. Today Examine the two ponds Collect egg clusters
Estimate population sizes (N)
= (# egg clusters) * 2