Allelic Frequency Changes Over Time in the Bean Beetle
Instructor Notes
Experimental Design
Any treatment students can think of can potentially skew the allelic frequency, for example
population bottleneck, heat treatment, cold treatment, changing bean host types for the
beetles, Raid spray. The important thing is to get the students to think about why and how
the allelic frequency may change.
Bean beetles can be frozen and DNA can be extracted later.
Populations (strains) of bean beetles obtained from many sources are homozygous for
many if not all genes. Before attempting this experiment, take at least two different strains
and cross breed to form a new heterozygous population.
Equipment and Supplies
For a class of 24, one single experiment could be done to keep costs down. Each PCR will
require a single bean beetle to have its DNA extracted, so to do the experiment correctly, a
class would need to buy a Qiagen DNAeasy kit with 250 preps. The PCR Platinum Supermix
has enough reagent for 100 reactions. To do this experiment correctly, at least 25 bean
beetles should be tested before treatment, although more is probably better. Then we
tested a few beetles after the treatment, then at least 25 after allowing the population to
live for a few generations. If each group wanted to go their own treatment, many kits and
PCR Supermix would need to be purchased.
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Bean Beetle cultures
Bean Beetle Bottles
Beans
Materials for student designed treatment
Minipestles
Microfuge tubes
Qiagen DNeasy Blood and Tissue Kit
PBS
PCR Primers P7 set
Invitrogen PCR Platinum Supermix
PCR Machine
PCR tubes
Gel electrophoresis apparatus
Agarose to prepare 1.9% gels
TBE or TAE (instructor preference)
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Ladder DNA for gel reference Bioexpress Gene-Mate Quanti Marker 1kb
DNA stain (Gel Red preferred)
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Phosphate Buffered Saline (PBS)
8 g of NaCl
0.2 g KCl
1.44 g of Na2HPO4
0.25 g of KH2PO4
Bring up to about 900 ml and pH to 7.0. Bring to 1 L, and autoclave.
PCR Primers
p7 is 5’AGGTTGCAGAAGGAGGCTCT3’ forward and 5’CCGTCCTGGAGCCATATCT3’
backward. This primer pair amplifies a short tandem repeat (STR) that was identified by
Smith and Hicks (2012) in their search for polymorphic loci in bean beetles. We bought
them from IDT and resuspended them in mpwater at 1g/l.
Other PCR primers were evaluated but were not acceptable. The primers mentioned below
all amplified but the loci they amplified were not clearly Mendelian in their inheritance.
For example, two primers for esterases (Raja et al 2009) amplified and indicated different
alleles in two populations but hybrid offspring did not show the expected heterozygous
genotype products. RAPD primers described by Fleurat-Lessart and Pronier (2006) (for
example P5 in Figure 3 below) amplified but the alleles were not inherited as expected, and
other RAPD primers (Gill et al 2006) amplified but were difficult to interpret as alleles in a
Hardy-Weinberg equilibrium analysis.
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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,
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.
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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.
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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).
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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 Tables
Pre-treatment Beetles:
350kb+
350kb
450kb
18
5
450kb
1
350kb +
200kb
0
450kb +
200kb
0
200kb
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Post-Treatment Beetles (note students didn’t check very many because they needed the
beetles for the rest of the experiment):
350kb+
350kb
450kb
350kb +
450kb +
200kb
450kb
200kb
200kb
5
0
0
0
0
2
5 Generations Post-Treatment:
350kb/
350kb
450kb
450kb
0
0
0
350kb +
200kb
0
450kb +
200kb
0
200kb
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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
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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
p2+2pq+q2+2pr+2qr+r2=1
Observed
p2=0.16
2pq=0.56
q2=0.03
2pr=0
2qr=0
r2=0.25
sum
18
5
1
0
0
8
32
Expected
p2=0.19 0.19(32) =
2pq=0.27
q2=0.10
2pr=0.22
2qr=0.16
r2=0.06
sum
Expected Number
6.08 ~ 6
8.64 ~ 9
3.02 ~ 3
7.04 ~ 7
5.12 ~ 5
1.92 ~ 2
32
Chi-squared analysis:
(18 − 6)2
(5 − 9)2
(1 − 3)2 (0 − 7)2 (0 − 5)2 (8 − 2)2
+
+
+
+
+
= Χ2
18
9
3
7
5
2
8+1.78+1.33+7+5+18=41.11
X2=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, Dmax=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.
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Literature Cited
Fleurat-Lessard, F. and Pronier, V. 2006. Genetic differentiation at the inter- and intraspecific level of stored grain insects using a simple molecular approach (RAPD).
Proceedings of the 9th International Working Conference on Stored-Product Protection
PS5-9 – 6305:446-455.
Gill, T.k., Kumri, S., Sharma, V.L., Badran, A.A., Kumari, M. and Sobit, R.C. 2006. Genetic
variation in polymorphic males of Callosobruchus maculatus (Coleoptera: Bruchidae) by
RAPD-PCR. Cytologia 71(1):57-62.
Raja, M., William, S.J., and Hussain, K.J. 2009. Genetic diversity of Callosbruchus maculatus
(Fab.) (Coleoptera: Bruchidae) populations. International Journal of Integrative Biology
8(1):15-18.
Smith, J. and K. Hicks. 2012. Detecting genetic polymorphisms in different bean beetles
populations. www.beanbeetles.org/protocols
This experiment was written by Pamela A. Marshall and Lara Ferry (Beanbeetles.org).
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