INTRODUCTION
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
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).
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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 F
1
progeny obtained is heterozygous for the mutation (+/-) and has a chromosome of the Ler ecotype and a chromosome of the Col ecotype. An F
1
plant is allowed to self-fertilize. The resulting F
2
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 F
2
are made of a mixture of the two ecotypes (Ler and Col).
Figure 1. Development of the F
2
plants needed to test linkage when mapping with CAPS markers. The star indicates that the gene of interest is mutated at an arbitrary position.
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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 F
2
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 g4026a
84.9 cM
Chr 1
135 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.
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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: r =
Number of Col/Ler + 2 X Number of Col/Col
2 X Number of plants analyzed
X 100
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) ]
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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 Beads TM
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 MgCl
2
, 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.
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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.
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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.
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Procedure I: Isolating DNA From Arabidopsis thaliana ago1 mutants
Reagents Equipment & Supplies Shared Items
Edward's Extraction Buffer,
400
 l
1.5 ml test tube, polypropylene
Isopropanol, 400
 l 100100 µl micropipet and tips
Tris/EDTA (TE) Buffer, 40
 l 1- 20
 l micropipet and tips
Microcentrifuge
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 F
2 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 1-
2 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.
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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
Pre-lab Preparation
1- 20
 l micropipet and tips Thermal cycler
 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.
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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
55
72



C for 30 sec.
C for 30 sec.
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.
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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
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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
UFOa g4026a
H77224a
TaqI
RsaI
TaqI cuts at 65

C cuts at 37

C cuts at 65

C
(no BSA) add BSA
(no BSA) 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.
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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:
Col Ler m235a
UFOa g4026a
H77224a
309 + 225 bp
983 + 316 bp
650 bp
130 + 90 bp
534 bp
600 + 383 + 316 bp
800 bp
130 + 70 + 20 bp
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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: r =
Number of Col/Ler + 2 X Number of Col/Col
2 X Numner of plants analyzed
X 100
Look at all the results the class obtained and evaluated the percentage of recombination of each CAPS marker with the AGO1 gene.
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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.
Chr 1
135 cM m235a
31.9 cM
UFOa
47.5 cM g4026a
84.9 cM
H77224a
113.2 cM
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