1. Map-based cloning of interesting genes
In a model organism
Generate mutants by mutagenesis of seeds
Use a genetic background with lots of known polymorphisms
compared to other genotypes. Availability of polymorphic
markers for mapping.
2. Select mutants with phenotypes of interest eg. Hairless or Glabrous
3. Clean up mutant genotype by backcross to wild type
all F1 will be heterozygous, mutant phenotype will
be either dominant or recessive.
4. Allelism tests with mutants that look similar
5. Select F2’s that are homozygous for the mutation again.
These can be used to map the location of the mutation

2. GLABROUS1 (GL1) Involved in trichome initiation Transcription factor
Expressed in leaf primordia  early trichome initiation
gl1 mutants result in near complete loss of trichome initiation
Wild type gl1 mutant

3. Scanning EM picture of Arabidopsis top leaf surface
with trichomes

4. Parents: Col-0 gl/gl X La-er GL1/GL1 genotypes
F1 is self fertilized –
all chromosomes recombine in meiosis
F2 plants –
recombined chromosomes segregate
How to do this with an organism that cannot fertilize itself,
like a mouse?

5. Mapping an Arabidopsis gene
Analyze segregation data in an F2 population.
Both chromosomes have had the opportunity to become recombined in the F1 parent
To avoid confusion, we focus on one locus of interest.
We chose individuals that are homozygous for one allele at that locus, eg. a clear phenotype.
Closely linked markers will also be homozygous in the chosen individuals. As markers are farther away on the chromosome, more of the individuals will have two different parental alleles for the marker genes.

6. Interval mapping: Identify markers linked to the gene of interest
that define an interval on a chromosome.

7. Markers that define major regions of the
Arabidopsis chromosomes

8. F2s are selected as homozygous recessive gl1/gl1 by phenotype
eg. Scored for 5 markers
1, 2 are not linked to
GL1
13: 25: 12
C/C:C/L:L/L
Map distance is calculated as
#recombinant alleles/total X 100 cM
50% of alleles are C and 50% are L.
Therefore the map distance from
GL1 to 1 is 50 cm

9. Marker 1 from a previous year.
The first lane is the glabrous mutant (Columbia),
the second lane is a mixture of DNA from lane 1 and lane 3
The third lane is Landsberg
The rest are DNA from F2 plants

10. We established that gl1 is on Chromosome 3.
What do we do next?
Identify 2 markers on Chromosome 3
that must be on either side of gl1

11. 3, 4, and 5 are linked to GL1 Marker 3 is closest to GL1
Map distance is calculated as
#recombinant alleles/total X 100 cM
3 is 4/100 X100 cM from GL1 = 4 cm
4 is 30 cM
5 is 20 cM

12. Markers 3, 4 and 5 are linked to Gl1
Need to find another marker
on the opposite side of marker 3 to define
the interval that contains GL1.
gl1
gl1 3 3

13. There is a recombination event between marker 3 and gl1
Which of the other 2
linked markers is on
the opposite side
from marker 3?
Plants 3, 4, 5 and 10
are useful to identify
flanking markers

14. Marker 3 and 5
define an interval
of 24 map units
that must contain
gl1

15. Markers 3, and 5 flank GL1 Plant 3 is C/L at 3 L/L at 4 and C/C at 5
There has been no crossover between
GL1 and 5
And 5 is further away from GL1 than 3.
This means 3 and 5 define the interval
that contains GL1.
Plant 3 5
gl1 3
Col-0
La-er 4

16. We screen a larger number of F2 plants
We only look at plants that are heterozygous
for either 3 or 5
We use more markers between 3 and 5 on these
plants only to narrow in on gl1

17. Plants 3, 4 and 5 have recombination points within
the interval that defines the location of GL1
They will be useful for further mapping
Plant 4
Plant 5
Plant 3 is C/L at 3
L/L at 4 and
C/C at 5
Plant 4 is C/C at 3
C/C at 4 and
C/Lat 5
Plant 5 is C/L at 3
C/L at 4 and
Plant 3
gl1 3 4 5
gl1 3 4 5
gl1 3 4 5
gl1 3 4 5
gl1 3 4 5
gl1 3 4 5
Col-0
La-er

18. Identify more plants with recombination in the interval
We will screen more F2 plants to identify
those with a recombination on either side
of our chosen interval to narrow in on the
location of the GL1 gene
Generally we screen at least 1000 F2 plants

21. Chose new markers within your large mapped interval and
repeat recombination analysis using those markers.
Continue until markers show no recombination.
Collect more informative recombinants, if possible.
Define the interval as narrowly as is practical using more
markers and more recombinants.
Once you have defined a minimal interval,
identify DNA clones in an ordered library that carry your
Markers.

22. Once we have defined 2 markers flanking our interval that are physically
close enough, we start sequence analysis for point mutations.
MDF20
MYN21
BAC T22A kb insert
BAC sequence gives us a list of genes. ~20 in Arabidopsis.
GenBank annotation gives us a list of predicted genes
for each BAC from our ordered library.
Potential functions of the predicted genes are defined by
homology to other proteins.
Candidate genes can be chosen by predicted function
and expression pattern.

23. Expression pattern of genes in mapped interval
can help choose best candidate gene

24. Candidate genes can be PCR amplified from the
mutant and the sequence can be
compared to wild type.
When a mutation is identified,
we call that a candidate gene.
Transform mutant plant with the
wild type candidate gene
for complementation.

25. Alternatively, the entire BAC can be broken
into subclones.
Each subclone can be used to transform
the mutant plant.
If the BAC is made with wild type DNA,
subclones with the correct
gene in them will
complement the mutation.

26. Final confirmation
Sequence mutant and wild type – multiple mutant alleles needed to be convincing
Complement mutation by making a transgenic with the wild type copy of the candidate gene.

27. Plants 3, 4, 5, 8 and 10 are useful to find closer markers There is a
recombination event
either between 3 and gl1
or between 5 and gl1

28. Oultine of
experiments for
Map-based
cloning

30. Using closely linked Markers to identify a small interval
containing GL
Once we find
flanking markers
that are both on
the same BAC,
we are done with
mapping F2 plants.

31. Making a library of clones that represent the entire genome -
Putting them in order to represent the genome sequence

32. Identify a BAC clone that must include your gene
Find two flanking markers contained in a single BAC (large insert plasmid) clone.
Look at GenBank entry for that BAC clone to identify candidate genes between your flanking markers
Open reading frames,
mRNA (cDNA) clone already identified,
Predicted gene regions

33. Once we have defined 2 markers flanking our interval that are physically
close enough, we start sequence analysis for point mutations.
MDF20
MYN21
BAC T22A kb insert
BAC sequence gives us a list of genes.
Candidate genes can be PCR amplified from the mutant
and the sequence can be compared to wild type.
When a mutation is identified, we call that a candidate gene.
Sequence the same gene from more than one mutant to confirm or
Transform mutant plant with wild type gene for complementation.

34. Final confirmation
Sequence mutant and wild type – multiple mutant alleles needed to be convincing
Complement mutation by making a transgenic with the wild type copy of the candidate gene.

35. Finding a gene based on phenotype
1. 100’s of DNA markers mapped onto each chromosome – high density linkage map.
2. identify markers linked to trait of interest by recombination analysis
3. Narrow region down to a manageable length of DNA – for cloning and sequence comparison
4. Compare mutant and wild type sequences to find differences that could cause mutant phenotype
5. Prove that mutation is responsible for phenotype.