MOLECULAR MARKERS(SSLP Mapping)

Objective: to find the location of the ttg gene in chromosome 5 ofArabidopsis thaliana

To achieve the objective: map the ttg gene with respect to a molecularmarker

What is mapping?Mapping is the process of determining the locus of a gene in a particular chromosome.

The processes1. Calculate the frequency of crossing over between the genes ofinterest: create a genetic map.

2. Scan the chromosomes of interest with chromosomal deletions and/orduplications: create a cytological map(limited to the organisms that have polytene chromosomes).

3. Find a linkage between the gene ofinterest and a molecular marker:create a physical map.

Note: There is no direct correlation between genetic, cytological, andphysical map, because the parameters used to construct the mapsdiffer in each technique.

How accurate is the genetic map?

R

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tx

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Single crossing over

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tT

Chromatid formation

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Double crossing over

Chromatid formation

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All “parental” (NCO)Recombinants(CO)

Parental Parental(NCO) (NCO)

Tt

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Replication during meiosis

White-shortRed-Tall

How to map a gene with great accuracyUse microsatellites!

Microsatellites are short DNA fragments in the genome that arerepeated several times in tandem:

………GTACGGTCATATATATATATATATCTTACGAA………

Miocrosatellites show polymorphism in different ecotypes. Forexample, the microsatellite nga162 is AT-rich:

nga162 in Col………………………...(AT)50nga162 in Ler…………………………(AT)10nga162 in Ws…………………………(AT)8

Because of this variability, they are called Simple Sequence LengthPolymorphism (SSLP).

tandem repeat

Advantages of using microsatellites1. Variation in the number of tandem repeats can be detected by

agarose gel electrophoresis.

2. Microsatellites are codominant: heterozygotes can be identified by virtue of containing both alleles.

How to detect microsatellites1. Extract DNA from the organism of interest.

2. Amplify the microsatellite of interest by PCR.

3. Electrophorese the amplified DNA.

4. View the gel.

How to detect microsatellites1. Extract DNA from the organism of interest.2. Amplify the microsatellite of interest by PCR.3. Electrophorese the amplified DNA.4. View the gel.

DNA DNA DNA

Gel

Col Ws Col/Ws

How to map a gene with respect to a microsatellite1. Generate a mapping population.

F2

WT

ttg WT(Ws bkgd) (Col bkgd)

XP

F1

CO

F1XF1

X X X

No CO CO in either parent CO in both parents

F2ttg

F2 is the mapping population

2. Extract DNA from the F2 ttg plants.3. Extract DNA from Col and Ws plants and use as control.4. Amplify the extracted DNA with respect to the microsatellite of

interest.5. Electrophorese the amplified DNA.6. On the gel compare the ttg bands with the controls to identify

crossovers and non-crossovers.

Gel

Col Ws ttg ttg ttgcontrol control NCO CO CO

Procedure1. In this exercise, bench 1 is Group 1 and bench 2 is Group 2.

2. Group 1 will use the microsatellite nga151.Group 2 will use the microsatellite SO262.

3. Location of the microsatellites is “unknown” to students.

4. Each group will extract ttg DNA from five ttg plants. Col and WsDNA will be provided.

-Place two leaves in each PCR tube-Add 40 µL 0.25 M NaOH, smash leaves with pipette tip-Heat the tube to 100ºC for 1 min-Place the tube on ice for 3 min-Add 40 µL 0.25 N HCl-Add 20 µL 0.5 M Tris-Mix the content by pipetting up and down-This is the DNA needed in step 5

Group 1 Group 2dH2O………………………80 μL dH2O…………...…………80 μLSSLP buffer……...……….20 SSLP buffer………………20MgCl2……………………..18 MgCl2………………..……18dNTP…………………...…20 dNTP……………...…..…..20nga151-F……………...….25 SO262-F………………….25nga151-R…………….......25 SO262-R………………….25Taq………………………….2 Taq…….……………………2

Distribute in 7 PCR Distribute in 7 PCRmicrotubes: 19 μL microtubes: 19 μL

in each tube in each tube

1 2 3 4 5 6 7 1 2 3 4 5 6 7

To tube #1 add 1 μL Col DNA To tube #1 add 1 μL Col DNATo tube #2 add 1 μL Ws DNA To tube #2 add 1 μL Ws DNATo tube #3 add 1 μL ttg DNA To tube #3 add 1 μL ttg DNATo tube #4 add 1 μL ttg DNA To tube #4 add 1 μL ttg DNATo tube #5 add 1 μL ttg DNA To tube #5 add 1 μL ttg DNATo tube #6 add 1 μL ttg DNA To tube #6 add 1 μL ttg DNATo tube #7 add 1 μL ttg DNA To tube #7 add 1 μL ttg DNA

5. Each group will prepare one reaction (master mix) in a 0.6 mLmicrotube. This reaction is enough for 10 PCR tubes.

6. Program the thermal cycler as described in Table 14-1 of the LabManual.

7. Place the tubes in the thermal cycler.

8. Next week electrophorese the PCR product to see the results.

To electrophorese: load the gel from left to right as indicated below:

Group 1 5 µL DNA ladder15 µL content of Col15115 µL content of Ws15115 µL content of each tube marked ttg151

Group 2 5 µL DNA ladder15 µL content of Col26215 µL content of Ws26215 µL content of each tube marked ttg262

Electrophorese for 45 min at 90 volts.

One of the many possible results

Top: no crossing overBottom: crossing over