Sample Data
These sample data were collected by
Bentley J. Hanish, Matthew Lam, Keeley Lee, Braelen D.
Svoboda, students at Arizona State University at the West Campus. In this experiment, bean beetles were exposed to high temperature (53°C) for 30 minutes. Much longer treatment kills the beetles.
Figure 1. Gel electrophoresis of bean beetle DNA showing PCR products of the polymorphic locus amplified by the P7 primers. The arrows indicate possible alleles amplified by the P7 primers. Each lane is an individual beetle from the parental generation, the LB beetle population.
1

Figure 2. Gel electrophoresis of bean beetle DNA amplified with P7 primers. DNA was extracted from individual beetles and amplified with P7 primers. Lane A1, Africa parental population. Lanes A2-A6, F2 generation of a cross between the LB and Africa population.
2

Lanes 10-14, bean beetle DNA of surviving beetles after 53°C treatment. Arrows indicate three alleles (amplification product sizes).
1000
700
500
200
Figure 3. Representative gel of PCR Analysis with P7 primers of individual beetles 5 generations after the heat treatment. The population seems to have gone to fixation with the 200kb allele (arrow).
3

Data Analysis
Usually a chi-squared test is used to determine whether a population is in Hardy-Weinberg equilibrium. Calculating the observed allele and genotype frequencies and expected frequencies with H-W equilibrium are explained in the Student Handout.
Using the P7 primer, we analyzed populations of beetles that survived the heat treatment.
We saw that the starting P generation either had a genotype of a ~450kb/~350kb, heterozygote or homozygous ~450kb or homozygous ~350kb from the LB population or a single ~200kb allele from the Africa population. After mating the two populations, we saw that the ~200kb was sometimes seen as well in individuals with the heterozygous or homozygous genotype. After treating these beetles with heat, many died, but those that lived, we allowed them to live for about 5 generations (a semester) and then sacrificed them, extracted DNA, and performed PCR with the P7 primers. We saw in all cases that the genotypes of the remaining beetles was only the ~200kb allele.
Data Tabulation
The raw data from which these tabulations were made is provided in the sample data spreadsheet available for downloading.
Pre-treatment Beetles:
350kb+
450kb
18
350kb
5
450kb
1
350kb +
200kb
0
450kb +
200kb
0
200kb
8
Post-Treatment Beetles (note students didn’t check very many because they needed the beetles for the rest of the experiment):
350kb+
450kb
350kb 450kb 350kb +
200kb
450kb +
200kb
200kb
0 0 2 5
350kb/
450kb
0 0
5 Generations Post-Treatment:
350kb 450kb 350kb +
200kb
450kb +
200kb
200kb
0 0 0 0 0 20
The population seemed to have gone to fixation after the treatment.
Population Analysis via Hardy Weinberg
Tri-allelic Hardy-Weinberg: p is 350kb q is 450kb r is 200kb
4

Pre-treatment p + q + r = 1 p = [18 + 5(2)]/64= 0.44 q = [18 + 1(2)]/64= 0.31 r = [8(2)]/64= 0.25 p 2 +2pq+q 2 +2pr+2qr+r 2 =1
Observed p 2 =0.16
2pq=0.56 q 2 =0.03
2pr=0
18
5
1
0
2qr=0 r 2 =0.25 sum
Chi-squared analysis:
0
8
32
(18 − 6) 2 (5 − 9) 2
+
18 9
+
Expected p
2pq=0.27 q
2
2
Expected Number
=0.19 0.19(32) = 6.08 ~ 6
=0.10
2pr=0.22
2qr=0.16 r 2 =0.06 sum
(1 − 3)
3
2
+
(0 − 7)
7
2
8.64 ~ 9
3.02 ~ 3
7.04 ~ 7
5.12 ~ 5
1.92 ~ 2
+
5
32
(0 − 5) 2
+
(8 − 2)
2
2
= Χ 2
8+1.78+1.33+7+5+18=41.11
X 2 =41.11, df=5, p<0.0001, but two genotypes have expected values of less than 5, so the chi-squared test is not appropriate. A Kolmogorov-Smirnov One-Sample test is a better choice when any expected values are less than 5 (www.vassarstats.net). In this case, the observed distribution in the pre-treatment population is significantly different from the expected Hardy-Weinberg equilibrium (n=32, D max
=0.375, p<0.01).
Post-treatment p + q + r = 1
0 + 0 + 1 = 1 r=1, the population has been skewed to one allele at this locus.
This experiment was written by Pamela A. Marshall and Lara Ferry (Beanbeetles.org).
5