Genetic Mapping with CAPS Markers

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Genetic Mapping with CAPS Markers
Adapted from: “Arabidopsis Molecular Genetics, Course Manual”
Cold Spring Harbor Laboratory
And from: “EMBO COURSE, Practical Course on Genetics”
“Genetic and Molecular Analysis of Arabidopsis”
“Module 2: Mapping mutations using molecular markers”
http://www.cnrs-gif.fr/isv/EMBO/manuals/ch2.pdf
INTRODUCTION
It is often necessary to determine the genetic map position of a gene defined only by a
mutation. Map positions are useful for testing whether a mutation corresponds to a
previously identified gene, and are essential for map-based strategies of gene cloning.
Since
Alfred
Sturtevant’s
1913
mapping
experiments
with
Drosophila
(http://vector.cshl.org/dnaftb/11/concept/index.html), new mutations have been mapped
by linkage analysis. Determining the map position of a gene (as identified by its mutant
phenotype) consists basically of testing the linkage with a number of previously mapped
genes or “markers” that also provide a phenotype. Genetic maps are constructed
based on the principle that the frequency of recombination between genes decreases
as the distance between them decreases. The frequencies of recombination between
the gene of interest and the genes previously mapped allow the gene of interest to be
placed on the map.
However, markers for genetic mapping don’t necessarily have to be mutations that
cause phenotypic changes. They can also be variations in DNA sequences that are
detectable by molecular methods. In Arabidopsis thaliana, molecular markers exploit
the natural differences between distinct ecotypes (sub-divisions of species). For
instance, it has been estimated that the widely used Landsberg (Ler) and Columbia
(Col) ecotypes differ by approximately 0.5 to 1% at the DNA level. The local differences
or polymorphisms of the DNA sequence are due to point mutations, insertions or
deletions that randomly occurred in one ecotype and not in the other. These DNA
polymorphisms can be conveniently visualized by several methods.
In this exercise, the class will map the AGO1 gene of Arabidopsis thaliana through a
PCR-based detection of DNA polymorphisms called CAPS markers (Cleaved Amplified
Polymorphic Sequences) (Konieczy et al, 1993). AGO1 is located somewhere on
chromosome 1 and the mutation of both copies (homozygotes +/+) produces a dwarf
plant since the gene expression affects leaf, flower, and auxiliary meristem development
(Bohmert et al, 1998).
1
For CAPS mapping, a plant of a certain ecotype (i.e, Ler) that is homozygous for the
mutation ago1 (+/+ for the mutation) is crossed to a wild-type plant (-/-) of a different
ecotype (i.e., Col) (see Figure 1). The F1 progeny obtained is heterozygous for the
mutation (+/-) and has a chromosome of the Ler ecotype and a chromosome of the Col
ecotype. An F1 plant is allowed to self-fertilize. The resulting F2 progeny is composed
of plants that are homozygous wild-type (-/-; about ¼), heterozygous for the mutation
(+/-; about ½), and homozygous for the mutation (+/+; about ¼). Due to crossing-over
events during gamette formation, the chromosomes in the F2 are made of a mixture of
the two ecotypes (Ler and Col).
Figure 1.
Development of the F2 plants needed to test linkage when
mapping with CAPS markers. The star indicates that the gene of interest
is mutated at an arbitrary position.
2
We will take advantage of the mixture of ecotypes in the chromosomes of the F 2
progeny to evaluate the number of crossing-over events between different regions of
the chromosome and the gene AGO1 and thus to locate the gene. The F2 plants that
are homozygous for the mutation of interest (+/+), and thus showing the mutant
phenotype, will be used for mapping. Since both of their chromosomes contain the
mutation and the mutation was from a Ler background, the number of crossing-over
events is equivalent to the number of times the Col ecotype is found on the
chromosome.
There are many DNA sequence variations among Arabidopsis ecotypes, and since
these are also segregating in the cross, they can be used as genetic markers. Among
these variations, CAPS markers are very useful. They are found in sections of DNA
that contain a restriction site present in one ecotype, but not in another. We will use
four CAPS markers located along chromosome 1 (135 cM long) so we can identify all
the sections (see Figure 2):
m235a
31.9 cM
Chr 1
135 cM
g4026a
84.9 cM
UFOa
47.5 cM
H77224a
113.2 cM
Figure 2.
Schematic location of the CAPS markers that will be used on
chromosome 1 of Arabidopsis thaliana.
Note: The scientific community has generated a large number of CAPS markers.
A list is publicly available through The Arabidopsis Information Resource (TAIR)
at the URL: http://www.arabidopsis.org/aboutcaps.html.
The CAPS markers are detected using PCR amplification and restriction analysis. The
sections of chromosomes corresponding to the CAPS markers are amplified with
specific PCR primers (the product is the same size for all ecotype DNA). The amplified
DNA is then cut by a restriction enzyme. In the example of Figure 3, the enzyme cuts
twice in the Ler ecotype DNA and three times in the Col ecotype DNA. The results of
the restriction are detected by gel electrophoresis. The pattern of the bands will indicate
if the plant is homozygous for the allele from one ecotype (Ler/Ler), heterozygous
(Col/Ler), or homozygous for the allele from the other ecotype (Col/Col), at the position
of the CAPS marker.
3
Figure 3.
Assaying CAPS markers by agarose gel electrophoresis. In
this case, the diagnostic restriction enzyme cleaves the amplified fragment
at either two or three sites depending on the ecotype of Arabidopsis.
In this exercise, the recombination frequency (r) between a particular CAPS marker and
the gene of interest is proportional to the number of chromosomes that are Col at the
CAPS marker. Its value in % is obtained by the following formula:
Number of Col/Ler + 2 X Number of Col/Col
r =
X 100
2 X Number of plants analyzed
It is necessary to convert the recombination frequency (in %) to a map distance (D, in
cM). In Arabidopsis, a reasonable estimate of map distance is given by the Kosambi
function:
D = 25 x ln [ (100 + 2r) / (100 – 2r) ]
4
EXPERIMENT: Mapping the AGO1 gene
The CAPS mapping experiment of the AGO1 gene can be broken into the following
steps:
I. Isolating DNA from Arabidopsis thaliana ago1 mutants using the Edward's
extraction protocol.
II. Amplifying the different CAPS marker locus by PCR.
III. Analyzing the PCR by gel electrophoresis to confirm amplification of DNA and
the yield.
IV. Cutting the DNA amplified by PCR with restriction enzymes.
V. Analyzing the restriction digests to identify the number of cuts that occurred and
thus the ecotype of that section of the chromosome.
VI. Compiling the results of the four CAPS markers to locate the AGO1 gene on a
map of chromosome 1.
Note on Amplifying DNA by PCR
Ready-To-Go PCR BeadsTM
Each PCR bead contains reagents so that when brought to a final volume of 25 l the
reaction contains 1.5 units of Taq polymerase, 10 mM Tris-HCl (pH 9.0), 50 mM KCl,
1.5 mM MgCl2, 200 M of each dNTP.
Primer Mix
This mix incorporates the appropriate primer pair (0.67m/l) in water. Loading Dye will
be used only before putting a part of the sample on agarose gel.
Setting Up PCR Reactions
The lyophilized Taq polymerase in the Ready-To-Go PCR Bead becomes active
immediately upon addition of the primer/loading mix. In the absence of thermal cycling,
“nonspecific priming” allows the polymerase to begin generating erroneous products,
which can show up as extra bands in gel analysis. Therefore, work quickly, and initiate
thermal cycling as soon as possible after mixing PCR reagents. Be sure the thermal
cycler is set and have all experimenters set up PCR reactions at the same time. Add
primer/loading dye mix to all reaction tubes, then add each student template, and begin
thermal cycling immediately.
5
To insure maximum specificity, some experimenters employ a "hot start" technique
where one reagent is withheld from the reactions until the samples are cycled to the
initial denaturing temperature. You can perform a “hot start” by adding the DNA
template during the first denaturation step. To program a “hot start”, one can extended
first denaturation of 10 minutes, or stop cycling and restart after adding template. A
simpler alternative is to set up reactions on ice, start the thermal cycler, and then place
the tubes in the machine as the temperature approaches the denaturing set point.
Cresol Red Loading Dye
This loading dye can be incorporated in the Primer mix when no restriction analysis is to
follow the PCR amplification. In this case, it will be added only to the part of the sample
that will be put on agarose gel.
Note on Gel Electrophoresis Analysis
Loading and Electrophoresing Samples
The object in these experiments is to let students determine either the level of DNA
amplification (and how much DNA to use for the restriction step) or the ecotype of the
chromosomes at the CAPS marker site. In the last case, the students will use the
information to evaluate the location of the gene of interest. It pays to load as many
samples possible on each gel in adjacent wells to help compare the results as a group.
However, analysis and sorting out anomalies will be greatly aided by adding at least one
lane of markers per row.
Some of the PCR products for the CAPS markers and some of the restricted fragments
are easily resolved on agarose mini-gels. This means you can double comb most minigel boxes with one set of wells at the top of gel and one set in the middle. But this is not
true for all of them. Here is a table suggesting when to use single comb or double comb
gels according to the CAPS being tested.
CAPS marker used:
For PCR
Amplification Analysis
For Restriction
Digest Analysis
m235a
UFOa
g4026a
H77224a
Double Comb Gel
Double Comb Gel
Double Comb Gel
Single Comb Gel
Double Comb Gel
Double Comb Gel
Single Comb Gel
Single Comb Gel
Since some of the DNA fragments will be very small, it is easier to pour agarose gels
containing ethidium bromide.
6
Cresol Red Loading Dye
The cresol red and sucrose functions as loading dye. Only a few microlitters are needed
with the sample you wish to put on the gel. The cresol red should not interfere with the
visualization of the bands of DNA. However, since it has relatively little sugar and
cresol red, this loading dye is more difficult to use than typical loading dyes. So
encourage students to load very carefully.
DNA Size Markers
For this experiment we favor a size marker that provides many bands at low sizes. A
good example of such a DNA size marker is the “100 bp ladder” sold by New England
Biolabs. This marker gives a band every 100 bp between 100 and 1,000 bp and two
more bands at 1,200 and 1,517 bp.
Viewing and Photographing Gels
View and photograph gels as soon as possible. Over time, PCR products disappear
from stained bands as they slowly diffuse through the gel.
7
Procedure I: Isolating DNA From Arabidopsis thaliana ago1 mutants
Reagents
Equipment & Supplies
Shared Items
Edward's Extraction Buffer,
400 l
Isopropanol, 400 l
Tris/EDTA (TE) Buffer, 40 l
1.5 ml test tube, polypropylene
Microcentrifuge
100-100 µl micropipet and tips
1- 20 l micropipet and tips
Disposable pellet pestle
Pre-lab Preparation


Plant Arabidopsis seeds and allow for a 3-4 week growth period. For information
concerning growing Arabidopsis, refer to The Arabidopsis Information Resource
(TAIR) at www.arabidopsis.org.
For each team of students, prepare aliquots of Edward’s Extraction buffer (400 l),
Isopropanol (400 l) and TE buffer (40 l).
Procedure
1. Grind tissue from an F2 ago1 (+/+) plant in a microfuge with plastic pestle for 1
minute. Note: Whole plants, single rosette leaves, single cauline leaves, whole and
partial influorescences have all worked. However, best results are obtained using 12 whole leaves.
2. Add 400 l of Edward's Extraction Buffer.
3. Grind briefly (to remove tissue from pestle).
4. Vortex 5 seconds; leave at room temperature for 5 minutes.
5. Microfuge for 2 minutes.
6. Transfer 350 l of supernatant to a fresh tube (quality is better than quantity at this
point).
7. Add 350 l of isopropanol, mix, leave at room temperature for 3 minutes.
8. Microfuge for 5 minutes, decant, air dry pellet for 10-15 minutes.
8
9. Resuspend DNA pellet in 100 l of TE Buffer taking care to resuspend the DNA that
might be on the side wall of the tube.
10. Template DNA can be used immediately or stored at -20C.
Procedure II: Amplifying the different CAPS marker locus by PCR.
Reagents
Equipment & Supplies
Shared Items
Primer Mix for each CAPS marker
(4), 22.5 l
Arabidopsis DNA, 2.5 l
Ready-to-Go PCR Beads (in
reaction tube)
Mineral oil (depending on thermal
cycler used
1- 20 l micropipet and tips
Thermal cycler
Pre-lab Preparation

For each team of students, you may want to pre-dissolve four Ready-to-Go PCR
beads with 22.5 l of each CAPS marker Primer Mix. In this case, skip step one
from the procedure.
Procedure
1. Use a micropipet with a fresh tip to add 22.5 l of CAPS marker primer mix to a PCR
tube containing a Ready-To-Go PCR Bead. Tap tube with finger to dissolve bead.
Make sure to label the tubes to know which CAPS marker will be amplified.
2. Use fresh tip to add 2.5 l of Arabidopsis DNA (from Part I) to each reaction tube,
and tap to mix. Pool reagents by pulsing in a microcentrifuge or by sharply tapping
tube bottom on lab bench.
3. Add one drop of mineral oil on top of reactants in the PCR tube. Be careful not to
touch the dropper tip to the tube or reactants, or subsequent reactions will be
contaminated with DNA from your preparation. Note: Thermal cyclers with heated
lids do not require use of mineral oil.
9
4. Label the cap of your tube with a number or initials to identify your team’s tubes.
Alternatively, note down the position where your tubes are placed in the thermal
cycler.
5. Store all samples on ice or in the freezer until ready to amplify. Program thermal
cycler for 30 cycles according to the following cycle profile. The program may be
linked to a 4°C to hold samples after completing the cycle profile, but amplified DNA
also hold well at room temperature.
Denaturing Time –Temp
Annealing Time – Temp
Extending Time – Temp
94C for 30 sec.
55C for 30 sec.
72C for 1min 30 sec.
6. Store the DNA amplified through PCR at 4°C until you are ready for the gel analysis
and the enzymatic restriction.
Procedure III: Analyzing PCR by gel electrophoresis to confirm amplification of
DNA and the yield.
Reagents
Equipment & Supplies
Shared Items
2% agarose gels with ethidium
bromide
1 X electrophoresis buffer (TBE)
100 bp ladder
1 – 20 l micropipet and
tips
1.5 ml test tubes
Electrophoresis
chambers
Power supply
Amplified DNA (from step II)
Cresol Red Loading dye (four x 1 l)
Pre-lab Preparation


For each team of students, prepare four aliquots of Cresol Red Loading Dye (1 l) in
test tubes.
You may want to pour the agarose gels containing ethidium bromide yourself before
the class starts. You may also place them in the electrophoresis chamber
beforehand so you are the only person handling the ethidium bromide. Each box
can be identified according to which CAPS marker will be analyzed in it.
10
Procedure
1. Transfer 5 l of the PCR of each CAPS marker in a tube containing 1 l of cresol red
loading dye. Identify each tube with the name of the CAPS marker as you go. Put
the PCR back at 4°C.
2. Use a micropipet with a fresh tip to transfer the 6 l of sample/loading dye mixture
into your assigned well of a 2% agarose gel. (IMPORTANT: Expel any air from the
tip before loading, and be careful not to push the tip of the pipet through the bottom
of the sample well).
3. Load 3 l of the “100 bp ladder” into one lane of gel.
4. Electrophorese at 140 volts for 20-30 minutes. Adequate separation will have
occurred when the cresol red dye front has moved 25 mm from the wells for double
comb gels and at least 40 mm for single comb gels.
5. Visualize the results. You are expecting the following bands: for m235a a band at
534 bp, for UFOa a band at 1300 bp, for g4026a a band at 900 bp and for H77224a
a band at 220 bp.
6. Determine the yield of the DNA amplification and how much should be use in the
next step. For a good amplification (bright band) 5 l of amplified DNA can be used
for the restriction analysis. For weak amplification (faint band) up to 10 µl of
amplified DNA can be used for the restriction analysis.
Procedure IV. Cutting the DNA amplified by PCR with restriction enzymes.
Reagents
Equipment & Supplies
Shared Items
Restriction buffer for each enzyme 1 – 20 l micropipet and tips Water bath at 37C
BSA solution
Water bath at 65C
Amplified DNA (from step II)
Restriction enzymes
Water
11
Pre-lab Preparation

For each team of students, you may prepare ahead the four restriction reactions
(“reaction mix”) by mixing restriction buffer, BSA (if needed for the enzyme), water
and restriction enzyme. This will help you save on the amount of enzyme used. The
enzymes to use with each CAPS marker are:
m235a
HindIII
cuts at 37C
(no BSA)
UFOa
TaqI
cuts at 65C
add BSA
g4026a
RsaI
cuts at 37C
(no BSA)
H77224a
TaqI
cuts at 65C
add BSA
Procedure
1. Make sure you know how much amplified DNA of each CAPS marker you should
use for the restriction analysis: 5 l with a good amplification and up to 10 l for a
weak one.
2. Evaluate the volume of water you should use to obtain 10 l total when added to
your volume of amplified DNA.
3. Add the volume of water you determined to each Reaction Mix tube (5 l or less).
4. Transfer the volume of amplified DNA determined from each CAPS marker DNA
amplification to the appropriate Restriction Mix tube (5 l or more).
5. Place your tubes in the appropriate water bath (37C or at 65C) for at least 2-3
hours.
6. The digestions can be stored at 4C until you are ready to do the gel analysis.
12
Procedure V. Analyzing the restriction digests to identify the number of cuts that
occurred.
Reagents
Equipment & Supplies
Shared Items
2% agarose gels with ethidium
bromide
1 X electrophoresis buffer (TBE)
100 bp ladder
1 – 20 l micropipet and
tips
1.5 ml test tubes
Electrophoresis
chambers
Power supply
DNA restriction (from step IV)
Cresol Red Loading dye
Pre-lab Preparation

You may want to pour the agarose gels containing ethidium bromide yourself before
the class starts. You may also place them in the electrophoresis chamber before
hand so you are the only person handling the ethidium bromide. Each boxe can be
identified according to which CAPS marker will be analyzed in it.
Procedure
1. Add 4 l of cresol red loading dye to each restriction digest. Make sure to change
tips each times.
2. Use a micropipet with a fresh tip to transfer the 20 l of sample/loading dye mixture
into your assigned well of a 2% agarose gel. (IMPORTANT: Expel any air from the
tip before loading, and be careful not to push the tip of the pipet through the bottom
of the sample well).
3. Load 3 l of the “100 bp ladder” into one lane of gel.
4. Electrophorese at 140 volts for 20-30 minutes. Adequate separation will have
occurred when the cresol red dye front has moved 25 mm from the wells for double
comb gels and at least 40 mm for single comb gels.
5. Visualize the results. You are expecting the following bands:
m235a
UFOa
g4026a
H77224a
Col
309 + 225 bp
983 + 316 bp
650 bp
130 + 90 bp
13
Ler
534 bp
600 + 383 + 316 bp
800 bp
130 + 70 + 20 bp
UFOa
m235a
g4026a
H77224a
Procedure VI. Compiling the results of the four CAPS markers to locate the AGO1
gene on a map of chromosome 1.
Procedure
1- The recombination frequency (r) between a particular CAPS marker and the gene of
interest is proportional to the number of chromosomes that are Col at the CAPS
marker. Its value in % is obtained by the following formula:
Number of Col/Ler + 2 X Number of Col/Col
r =
X 100
2 X Numner of plants analyzed
Look at all the results the class obtained and evaluated the percentage of
recombination of each CAPS marker with the AGO1 gene.
14
2- It is necessary to convert the recombination frequency (in %) to a map distance (D,
in cM). In Arabidopsis, a reasonable estimate of map distance is given by the
Kosambi function:
D = 25 x ln [ (100 + 2r) / (100 – 2r) ]
Convert the percent of recombination obtained between each CAPS marker and the
AGO1 gene into map distance. Use the following map to locate where the AGO1
gene is on chromosome 1.
m235a
31.9 cM
Chr 1
135 cM
g4026a
84.9 cM
UFOa
47.5 cM
15
H77224a
113.2 cM
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