Download Spring 2007 BIOL 212 General Genetics Eukaryotic Linkage

Spring 2007
BIOL 212
General Genetics
Eukaryotic Linkage Mapping Lab
Purpose:
To gain experience in setting up and analyzing genetic crosses in Drosophila. To
determine the order and map distances between genes in Drosophila by analyzing
recombination frequencies.
Reading:
Chapter 3, Hartl and Jones
Materials:
Drosophila wild type and mutant stocks
Drosophila culture medium
Fly vials and plugs
Fly Nap kits
Anesthetizers
Brushes and index cards
Dissecting microscopes
Carolina Drosophila manual
Procedure and time frame:
Prior to Week 1: Set up and maintain Drosophila stock cultures. Assist instructor at
pre-arranged times.
1. Week 1 (~Feb. 15-23): Observe and identify the two mutant traits. Collect double
mutant virgin females and wild type males, set up F1 crosses. This will be done mostly
outside of scheduled lab times; possible times will be announced in class and posted on
blackboard. Note: To obtain full credit for the lab, you will need to attend several of
these sessions.
 Questions you should prepare for (see Carolina Manual for answers to these and
many other questions):
o How do you distinguish male and female fruit flies?
o Why must virgin females be used in these crosses?
o How long can you leave the flies and be sure they are still virgins?
o How are Drosophila crosses set up?
2. Week 2: Remove P1 adult flies (by arrangement with instructor), once larval stages
are seen in the cultures. Prepare a flow chart or protocol outlining the steps involved in
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the crosses and giving the predicted genotypes and phenotypes of the flies involved,
using correct Drosophila symbols, due by Friday Mar. 9, 2007.
 Prepare to address these questions:
o What are the stages of the Drosophila life cycle?
o When should you expect to see larvae, pupae, newly hatched adults?
o How long does it take to complete the life cycle? Is there a risk of mixing
the generations?
o What genetic symbols are used in Drosophila publications for the traits
we are studying?
A suggested outline for the flow chart/protocol is shown in Figure 1. You should
research the fly mutations and work through what you think is the most likely hypothesis
to construct your own more applicable version of the flow chart/protocol.
________________________________________________________________________
Figure 1:
Drosophila linkage protocol
Name________________
Section_______________
[provide a brief introduction to the lab, introducing the traits and any research you have
done that suggests for each one, whether it is dominant or recessive and X-linked or
autosomal]
[Define traits and appropriate genetic symbols used; this example protocol is for two
autosomal dominant traits]
Trait 1: A = dominant phenotype 1
A+=wild type
Trait 2: B = dominant phenotype 2
B+=wild type
Crosses:
P1 cross: [Assume true-breeding stocks provided by instructor]
P1 cross: Female double mutants
AB/AB
X
male wild types
A+B+/A+B+
Punnett Square to illustrate P1 cross and F1 progeny produced:
A+B+
A+B+
AB
AB/A+B+
AB/A+B+
AB
AB/A+B+
AB/A+B+
2
F1 progeny predicted (genotypes and phenotypes):
F1 Females AB/A+B+
F1 Males AB/A+B+
double mutant phenotype
double mutant phenotype
F1 cross: [Carry out a testcross of F1 dihybrid females and males that are homozygous
recessive for both traits—in this example, these are male wild types]
F1
Females double mutant phenotype X Males wild type
AB/A+B+
X
A+B+/A+B+
Punnett Square to illustrate F1 testcross and predicted F2 progeny genotypes [assuming
independent assortment of genes]
AB
A+B+
AB+
A+B
A+B+
AB/A+B+
A+B+/A+B+
AB+/A+B+
A+B/A+B+
A+B+
AB/A+B+
A+B+/A+B+
AB+/A+B+
A+B/A+B+
phenotypes
Double mutant
Wild type
Mutant A phenotype
Mutant B phenotype
If genes are linked, then F2 progeny phenotypes will be mostly:
Double mutant males and females
Wild type males and females
A linkage map can then be constructed based on the F2 progeny results.
________________________________________________________________________
3. Week 3. Analyze the F1 flies to determine whether each of the traits are dominant or
recessive, X-linked or autosomal (in scheduled lab period or in out-of class time). Hand
in results from F1 analysis (in lab or as you complete them at other times). Collect virgin
F1 females (out of class time) and set up an F1 testcross with double mutant males.
 Questions to think through ahead of time:
o What is the outcome in the F1 generation of transmission of a dominant
trait from mother to offspring?
o What is the outcome of transmission of a recessive trait?
o How can you tell whether a trait is X-linked or autosomal?
4. Week 4: Remove F1 adult flies (done by instructor or outside of class).
5. Week 5: Observe and analyze F2 flies (in scheduled lab period or outside of class).
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6. Week 7: Analyze the F1 and F2 fly results and hand in your answers to the following
on separate pages (2 or 3 pages, typed or neatly hand-written, double spaced), due
byApril 24, 2007.
a. What is the most likely mode of transmission of the two traits, based on the F1 results:
autosomal dominant, autosomal recessive, or sex-linked recessive? Explain your answer.
b. Are the two genes linked or unlinked? Test hypothesis of independent assortment for
your F2 results (even if in your protocol, your hypothesis was complete linkage), using a
chi-square test (p=0.05). Provide your chi-square table and label as Table 1. State
whether you accept or reject your hypothesis, and what your conclusion is regarding
linkage.
c. If the genes are linked, construct a linkage map. Use the combined class data to
determine the order of the genes, calculate recombination frequencies, and construct a
linkage map. Label your linkage map as Fig. 1.
d. Based on the class results, are all the phenotypic classes represented proportionally to
what was expected. Why weren’t there more double mutant flies observed?
e. In a sentence or two, compare the results you find to published results. Be sure to
provide a complete citation of the book or article, or give the URL of web sites you
consult.
 Questions for thought:
o How do you determine whether genes are linked or unlinked? What
statistical test can be used to support linkage?
o How are testcross results analyzed to calculate recombination
frequencies?
Assignments:
1. Drosophila crosses and strain maintenance, 10 points. Participation in setting up and
maintaining Drosophila crosses will involve some out of class time (about 3 sessions), to
be arranged with the instructor, to be documented by sign-in sheets.
2. F1 results (5 points) and Protocol (15 points). For a suggested format for your
proposal, see Figure 1.
3. F2 results (5 points), Linkage map and analysis (15 points).
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