Transcript S1a. Genetic Linkage-Lesson Power Point Slidesx

Teaching Genetic Linkage and
Recombination through
Mapping with Molecular
Markers
Lisa McDonnell & Jennifer Klenz
Note for instructors: Please see the notes field for
comments that might be useful to you as an
instructor when running this lesson.
Mapping with
Molecular Markers
Where is the drought tolerance locus?
Lesson Plan
Part 1
• Assessing genetic linkage between a trait of interest
and a molecular marker if we do a testcross
(dihybrid F1 x homozygous recessive).
Part 2
• Predicting and assessing genetic linkage when we
do a dihybrid x dihybrid cross (F1 x F1)
Learning Objectives
• Explain how molecular markers (such as SNPs, microsatellites,
VNTRs) can be used to map the location of genes/loci,
including what crosses you would do, and why.
• From analysis of data from a cross (such as gel banding
patterns) determine if crosses involve linked genes.
• Be able to justify your analysis by describing the information in
the data that allows you to determine genes are linked.
• Use linkage information between multiple loci to construct a
genetic map
Brainstorm in your groups
 Genes closer together on a chromosome are more often
inherited together
1. If we know the location of some genes in a genome, how can
we use that information to determine what DNA sequences
cause specific traits of interest?
2. What are some examples of traits for which you (or someone)
might want to know the location of a gene in the genome that
controls that trait?
3. Why would you want to know the location of a gene?
Write down your group answers and hand them in
Using genetic linkage to map the
location of genes for traits of interest
“Drought Gene”
National Center for Atmospheric Research | University
Corporation for Atmospheric Research
Example: a drought tolerant plant.
Most plants do not thrive in drought conditions.
They may still reproduce, but they have lower yield.
Goals:
1) Is the drought tolerant phenotype heritable?
2) Is it caused by a single gene?
3) Mapping – where in the plant’s genome is the
mutation/locus involved in drought tolerance?
Plants are grown under drought conditions
Cross #1: Drought tolerant plants selfed:
x
Cross #2: Drought tolerant x true-breeding wild-type (drought sensitive)
x
Cross #3: offspring of cross #2
crossed to each other
x
What can you conclude
based on these results?
A. Wild type is dominant
B. Drought tolerance is dominant
C. Drought tolerance is heritable
D. A and C
E. B and C
We have a true-breeding drought tolerant population,
and we know drought tolerance is:
– recessive to wildtype (sensitive)
– caused by a single gene
What do we not know?
Goal: Map (identify) the location of the
locus/gene/mutation that causes the drought
tolerant phenotype.
• Wildtype is drought sensitive, DRS
• New mutant is drought tolerant, DRT
•
DRS is dominant to DRT
• We only know the phenotype and hypothetical
genotypes assigned based on the results of crosses:
F1:
X
DRT / DRT
DRS /DRS
DRT / DRS
Here is a map of known microsatellite markers in our plant’s
genome. How can we use these markers to find the locus
involved in drought tolerance?
Chromosome 1
2
3
4
5
122014 10:2; CC 4.0
Cosson et al. Plant Methods
How do we find the locus involved in drought tolerance
(DR)?
Here is a map of known microsatellite markers in our plant’s
genome:
Chromosome 1
DR locus?
2
3
5
4
DR locus?
D
F
A
C
DR locus?
B
E
G
DR locus? -
Does the drought tolerance phenotype segregate with
a the G microsatellite locus?
To measure segregation/linkage between 2 loci what do
we look for?
Frequency of parental and recombinant
combinations of genotypes/phenotypes.
For our experiment, this means segregation of
drought tolerance with a G microsatellite allele (a
specific banding pattern).
We need two “parental” combinations of alleles:
1) Drought tolerance with a G microsatellite allele
that gives one banding pattern
2) Drought sensitive with a different G
microsatellite allele that gives a different
banding pattern
G microsatellite
Image adapted from Cosson et al. Plant Methods 2014 10:2; CC 4.0
Is the drought tolerance locus linked to the G microsatellite?
Experimental plan: a testcross to measure linkage
1. Identify a true-breeding drought sensitive strain that carries
different microsatellite alleles (depending on the species these are
available for purchase form research groups)
2. Generate a heterozygous plant,
DRT G200 /DRS G400
3. Set up a testcross: heterozygote x homozygous recessive
4. Analyze offspring of the testcross, look for segregation of the
drought tolerance phenotype with G200 and G400 alleles.
Let’s start with the G microsatellite.
Is the DR locus genetically linked to G?
Microsatellite G
DR locus?
You have homozygous drought tolerant plants that have the 200
allele of the G microsatellite, and the drought sensitive plants are
homozygous for the 400 locus:
true-breeding:
drought tolerant
G200
DRT G200/ DRT G200
true-breeding:
drought sensitive
G400
DRS G400/ DRS G400
Is the drought tolerance locus linked to the G microsatellite?
Parent plants:
Drought tolerant
Drought sensitive
DRTG200 / DRTG200
DRS G400 / DRSG400
Ultimately we need to look for recombination (or
independent assortment) between the DR and G loci.
What crosses will we do next?
Generating a heterozygous plant
X
DRT G200 / DRT G200
F1
DRS G400 / DRS G400
• Draw chromosomes
of the parent plants
and the resulting F1.
• What phenotypes
will we see in the F1?
• What G microsatellite
banding patterns will
we see from the
parent and F1 plants?
Parent:
400
200
Parent:
F1 plant:
Expected F1 phenotypes and banding patterns
F1:
X
P generation:
DRT G200 / DRT G200
Parent:
drought
tolerant
400
200
DRS G400 / DRS G400
Parent:
Drought
sensitive
F1 plant:
drought
sensitive
DRS G400/ DRT G200
Assessing linkage: using a testcross
F1
x
homozygous recessive (“tester”)
DRS G400 / DRT G200
Drought sensitive
DRT G200 / DRT G200
Drought tolerant
F2
1. What gamete genotypes will each plant produce? (draw the
chromosomes!)
2. Which are parental? Recombinant?
3. What phenotype (drought tolerant or sensitive) and what
banding patterns will F2 have? (draw a Punnett Square to make
your predictions)?
4. What F2 results will suggest linkage between the DR locus and
G microsatellite locus?
Is the drought tolerance locus linked to the G microsatellite?
Which combination will go up if the DR and G
loci are genetically linked?
A.
B.
C.
D.
F2
1 and 3
1 and 4
2 and 3
2 and 4
Drought
tolerant
1
Drought
tolerant
2
Drought
sensitive
3
Drought
sensitive
4
400
200
CLICKER Question
F2
Drought
tolerant
Drought
tolerant
Drought
sensitive
Recombinant
Drought
sensitive
Parental
400
200
One of these is parental
One of these
22 is recombinant
Linkage: less than 50% recombinant types
• If two loci assort independently we expect 50% parental types
and 50% recombinant types.
• If two loci are linked we expect less than 50% recombinant
types
Frequency of recombinants = Number of recombinant types
Total number of samples analyzed
Is the drought tolerance locus linked to the G microsatellite?
Now that we’ve made predictions, let’s analyze
some data
Our testcross scenario:
F1 x homozygous recessive F2
Examine the four gels and determine which (if any)
indicates the drought tolerance locus is genetically
linked to the G microsatellite? (gels are on the next
two slides)
What is the map distance?
Which result indicates the drought tolerance locus is genetically linked
to the G microsatellite? (these are F2 results from the testcross)
Gel A
1
10 drought tolerant plants analyzed:
2
3
4
5
6
7
8
9
10
400
200
Gel B
1
400
200
2
3
10 drought sensitive plants analyzed:
4
5
6
7
8
9
10
Which result indicates the drought tolerance locus is genetically linked
to the G microsatellite? (these are F2 results from the testcross)
Gel C1
10 drought tolerant plants analyzed:
2
3
4
5
6
7
8
9
10
400
200
Gel 1D
400
200
2
3
10 drought sensitive plants analyzed:
4
5
6
7
8
9
10
Is the drought tolerance locus linked to the G microsatellite?
F1 x tester  F2
From your analysis of the four gels, which (if any)
indicates the drought tolerance locus is genetically
linked to the G microsatellite locus.
A.
B.
C.
D.
E.
Gels A & B
Gels C & D
Gels A & D
Gels B & C
None of the results indicate linkage
What is the map distance?
CLICKER Question
Summary
Always determine what allele combination is
considered parental, and which is recombinant.
Draw the chromosomes of the parents and F1 to
be sure you keep track of information correctly.
If you do an F1 testcross you can analyze the entire
F2 population to determine linkage. You always
know what gamete genotype the tester donates,
so the F2 phenotype proportions are
representative of the recombination events (and
frequencies) that happened in the F1.
Part 2:
Determining linkage when we do
a dihybrid cross
What if we do not cross the F1 to a
homozygous recessive plant, but instead
we let the F1 self?
DRT G200 / DRS G400
Drought sensitive
X
DRT G200 / DRS G400
Drought sensitive
DRS
G400
DRS
G400
DRT
G200
DRT
G200
Experimental Plan
Plants are grown under drought conditions
Selfed Many hundreds of F2 plants.
DRT G200 / DRS G400
Screen phenotypes & use PCR
for the microsatellite marker
Insert a picture of a
growth chamber,
such as the picture
found at:
http://www.rochmec
hatronics.com/Humid
ity-enviornmentalchamber.html
P generation:
X
DRTG200 / DRT G200
F1:
DRT G200 / DRS G400
Drought sensitive
Predict what the F2
data will look like if
there is linkage
DRS G400/DRS G40
Self-fertilized
Predict what the F2 data
will look like if there is
independent assortment
Prediction Activity
Students assemble into groups of 6 and then break into 2
mini-groups: 3 people in each mini-group.
3 people will
Each group, on the flip chart 3 people will predict
“predict what the
paper: draw gametes,
what the F2 will look
F2 will look like,
chromosomes with alleles
like, and banding
and banding
labelled, banding patterns on patterns, if marker
patterns, if marker a gel. Put your work on the and DR locus are not
and DR locus are
wall when you are done.
linked (assort
linked”
COMPLETE HANDOUT A
independently)
Class Slide 1
Come back together in your group of 6. Teach each
other about your predictions and identify how they are
different, and how they are different from when we
did an F1 x tester cross. COMPLETE HANDBOUT B.
F1x F1  F2
Consider the drought sensitive F2 plants
What is recombinant?
400
400
400
200
200
200
B.
C.
A.
D. B and C
E. Each can represent a recombinant
CLICKER Question
F1 self.
Drought tolerant F2 plants only
What is recombinant?
400
400
400
200
200
200
A.
B.
C.
D. B and C
E. Each contains a recombinant band
CLICKER Question
Which plants would you use to test for genetic linkage?
A. The entire F2 population
B. The F2 drought tolerant plants only
C. The F2 drought sensitive plants only
CLICKER Question
Based on these PCR results (banding patterns), what is
the linkage/map distance between the DR and G loci?
10 drought tolerant F2 plants analyzed:
1
2
3
4
5
6
7
8
9
10
400
200
A. Not linked
B. Linked but cannot determine distance
C. Linked, 20 map units apart
D. Linked, 30 map units apart.
CLICKER Question
How do we
calculate map
distance?
F1
x
homozygous recessive
DRT G200/ DRT G200
• Only gametes from the F1 can
contain recombinant genotypes
(chromosomes)
• Each F2 plant receives one gamete
from the F1, so only one “band” in
each F2 plant can be parental ore
recombinant.
• Count the number of
recombinant bands/total number
of plants
x
F1 self or F1 x F1
DRT G200 / DRS G400
• Gametes from each F1 can be
parental or recombinant.
• Both gametes that make the F
2plant come form the F1, so both
“band” in an F2 need to be
classified as either parental or
recombinant
• Count the number of
recombinant bands/total number
bands
X
G200 /G200
G400 /G400
Sensitive
Drought tolerant
Drought tol. Wilting
(parent))
(parent)
G200 /G400
F1: sensitive
F1 self-fertilized and F2
drought tolerant plants
analyzed.
F1
Drought tolerant F2 plants only
CONTROLS
The F2 banding pattern for all of the drought tolerant plants is the same as the
banding pattern shown above. Which is the best explanation for these F2 results?
The drought tolerance locus and the molecular marker are:
A. Genetically linked and some cross-overs occur between them.
B. Genetically linked and no cross-overs occur
C. Assort independently
D. This result is impossible.
CLICKER Question
Practice Assessing Linkage by analyzing results of an
F1 x F1 cross
New trait of interest: potatoes that are resistant to the
Colorado Potato Beetle.
a pest that destroys millions of dollars of potato
crops each year (and the beetle is resistant to
most insecticides)
© 2008, Pilise Gábor.
Creative Commons 3.0
You have found a potato plant
that potato beetles do not
attack. Is the plant producing
something that deters attack
from the beetle (or kills the
beetle)?
Goal: Find the locus involved in beetle resistance (BR)
2
Chromosome 1
5
4
3
E
C
F
A
D
B
We’ll call the locus involved in potato beetle resistance BR
BRR is the resistance allele
BRS is the wildtype allele (potatoes are susceptible to beetle attack)
BRS is dominant to BRR
We have six molecular markers we are using: A-F
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HANDOUT C
In your teams of 3, analyze the banding patterns and decide if
linkage is occurring between the BR locus and the microsatellite
markers.
F1s: Susceptible
Resistant plant X Susceptible plant
R A200/BRS A400
S A400/BRS A400
BR
R
200
R
200
BR
BR A /BR A
Marker Allele in
resistant
parent
plant
A
200
Allele in
susceptible
parent
plant
400
B
350
100
C
90
550
D
50
75
E
675
600
F
220
410
F1s self-fertilized
Which F2 plants will we
select for our analysis?
- Beetle resistant
- Normal
Use handouts with gel data from different markers to
determine the location
• Determine linkage
• Calculate map distance
• First group of 3 with the correct
answer wins bragging rights!
For your interest: What do you do next?
If the genome for this plant is sequenced: Look at the genes in
this approximate location. At a specific map location there
could be anywhere from 20-500 genes depending on the
genome.
Depending on the phenotype of interest it could be difficult or
easy to make educated guesses as to possible target genes.
A
B
BR, Beetle resistant locus
For species like Arabidopsis there are databases with additional
sources of helpful information such as whether any gene is
expressed under specific conditions, including attack by pests
such as the beetle. Genes in this region could be checked to see
what their wild-type expression pattern looks like under pest
attack.
You could compare the DNA sequence of your beetle resistant
plant to wildtype for your most likely candidate genes to look
for sequence differences causing the mutant phenotype.
You could take a wild copy of a likely candidate gene and add it
to the mutant to see if the wild type phenotype
is restored.
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