This document is a comprehensive lab manual for performing linkage mapping using MapMaker software, detailing steps for data preparation, execution, and analysis. It includes instructions for downloading necessary files, setting up the software, and processing sample datasets to build genetic linkage maps. Additionally, it provides guidance on grouping and ordering markers, as well as resources for graphical presentations of the results.

1. Lab Manual
Sameer Khanal
available at: https://www.slideshare.net/SameerKhanal1

2. Linkage Mapping using MapMaker
Software download site:
http://research.franklin.uga.edu/devoslab/scripts-used-genetic-mapping
Latest version: MapMaker/EXP 3.0b January 1993
Destination: closest to root file, normally ‘C’ in windows, works better
Executable file: MapMaker.exp (command prompt)
Citation:
Lander E.S., P. Green, J. Abrahamson, A. Barlow, M.J. Daly, S.E. Lincoln and L.
Newburg (1987). MAPMAKER: An interactive computer package for constructing
primary genetic linkage maps of experimental and natural populations.
Genomics 1(2):174-181.

3. Sample Data File for Linkage Mapping
Step 1. Go to
https://plant-breeding-genomics.extension.org/mapmaker-tutorial/
Step 2. Download “mapmakersampledataset.xls” (from
attachments), copy and paste
“mapmakersampledataset.txt” to notepad (save as same)
Save in folder where you would want to store the output
files
Data source:
Scott Wolfe (2012). MapMaker Tutorial. Web accessed: Aug 26, 2020 from:
https://plant-breeding-genomics.extension.org/mapmaker-tutorial/

4. Sample Data File for Linkage Mapping
Mapping file information a:
Total number of markers: 27
Mapping population: F2 intercross (Parent A x Parent B)
Total number of individuals: 104
Marker symbol:
- 1 = Parent A (homozygous for parent A alleles)
- 2 = Heterozygous (both parent A and parent B alleles)
- 3 = Parent B (homozygous for parent B alleles)
- 4 = Not homozygous for parent A
- 5 = Not homozygous for parent B
a https://pbgworks.org/sites/pbgworks.org/files/mapmakersampletextfile.txt

5. Sample Data File for Linkage Mapping
Step 3. Check input file format:
Source: https://pbgworks.org/sites/pbgworks.org/files/mapmakersampletextfile.txt
Type of Population
Population
Size
Number of
Markers
Defaults
Genotype Score
ScoresMarker Names

6. Start MapMaker
Step 4. Double click MapMaker.exe and run the program

7. Set Working Directory
Step 5. Change directory (cd command) to folder where your input
file mapmakersampledataset.txt is located

8. Upload Input File
Step 6. Upload the input file using prepare command
Here, prepare mapmakersampledataset.txt

9. Saving work
Step 7. Save occasionally to avoid loss of work. Use photo
command. Here, saved as “output1.out”.

10. Specify data
Step 8. Specify data to be used using sequence command. Here all
marker data is selected

11. Grouping
Step 9. Build preliminary linkage groups using group command.
Default thresholds are LOD = 3 and max. rf = 50
Two groups with
14 and 13
markers; no
unlinked markers

12. Grouping
Step 10. Check at different LOD and max. rf values. Here,
two groups remain unchanged at higher values.
One unlinked at
LOD =7 and max.
rf. = 30 (very
stringent values).
Back to original
grouping

13. Working on Group 1
Step 11. Specify the group (use seq) to start working on that
group. Here, start with the first group identified as group 1.

14. Ordering Markers in Group 1
Step 12. Linear order of markers in a specified group can be
obtained using order command
Automatic
Ordering steps:
1. Finds most
informative subset
and map them
2. Adds remaining
markers individually

15. Ordering Markers in Group 1
Step 12. Linear order of markers in a specified group can be
obtained using order command
Automatic
Ordering steps:
1. Finds most
informative subset
and map them
2. Adds remaining
markers individually
3. Tries unmapped
ones at lower
threshold

16. Ordering Markers in Group 1
Step 12. Linear order of markers in a specified group can be
obtained using order command
Automatic
Ordering steps:
1. Finds most
informative subset
and maps them
2. Adds remaining
markers individually
3. Tries unmapped
ones at lower
threshold
4. Reports markers
that do not fit
uniquely

17. Add Remaining Markers to Group 1
Step 13. First, seq order1 (best fitted group 1 markers). Then, add
remaining markers with try command. Remember, different
original subset could lead to different unassigned markers
Adding unassigned markers:
1. Try remaining markers. Start with
first one (marker 10 in this case)
2. Marker 10 best fits 3rd position

18. Update Group 1
Step 14. Make new sequence with additional marker at best fit
position, add remaining markers, and build final sequence
Adding unassigned markers:
3. Make new sequence with marker
10 added to 3rd position
4. Try other unassigned markers
sequentially
5. Make updated sequence

19. Finalize Linkage Group 1
Step 15. Finally, map command is used to build genetic linkage
map of the first group.

20. Linkage Group 2
Step 16. Repeat Steps 11 to 15 to build remaining linkage groups
(here, second linkage group)

21. Genetic Linkage Maps
MapChart a used for graphical presentation of genetic linkage map
a Source: https://www.wageningenur.nl/en/show/Mapchart.htm