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

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

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

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

Determining microsatellite alleles in an individual 1) Polymerase chain reaction (PCR) 2) Use (fluorescently-labeled) primers specific for flanking sequences [CATA]n http://www.dnalc.org/ddnalc/resources/pcr.html

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

Sample Fragment Analysis black, MS red, size standard

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

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)

Questions (pp. 91-96) 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

Questions (pp. 91-96) 2) Is there evidence of inbreeding in either subpopulation? Involves tabulating expected heterozygosity Remember… exp frequency heterozygotes = (1 - exp frequency of homozygotes)

Questions (pp. 91-96) 3) Is genetic drift a significant force driving divergence between the two subpopulations? Genetic drift = random divergence of allele frequencies between subpopulations

Questions (pp. 91-96) 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

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)

Rana tadpole sibling interactions aggregation repulsion Molecular Ecology (2006) 15, 1139-1145

Questions (pp. 91-96) 6) How do tadpole siblings position themselves relative to each other? Question 5 of next prelab asks you to design an experiment.

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