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Map-based cloning of interesting genes
In a model organism
1. 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 Glabra
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.
GLABRA1 (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
Scanning EM picture of Arabidopsis top leaf surface
with trichomes
Mapping Cross
Parents: Col-0 gl1/gl1 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?
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 alleles for the marker genes.
Interval mapping:
Identify markers linked to the gene of interest
that define an interval on a chromosome.
Markers that define major regions of the
Arabidopsis chromosomes
Marker 1 from a previous year.
The first lane is the glabra 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
If marker is unlinked to GL1, we expect ¼ L/L, ½ L/C, ¼ C/C
F2s are selected as
homozygous recessive
gl1/gl1 by phenotype
eg. Scored for 5 markers
marker
1
2
3
4
5
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.
Marker 3 is linked to GL1.
46 C/C: 4 C/L: 0 L/L
The map distance from GL1 to 3 is 4/100 X 100 cM or 4 cM.
We established that gl1 is on Chromosome 3.
What do we do next?
We can only find a locus by identification of
recombination events on either side.
Identify 2 markers on Chromosome 3
that must be on either side of gl1
www.arabidopsis.org
Tools
Mapviewer
Chr 3
Markers
Zoom to 200X
Clones
Markers 3, 4 and 5 are linked to GL1
on chromosome 3
We need to find another marker
on the opposite side of marker 3 to define
the interval that contains GL1.
5
gl1?
3
gl1?
4
There is a
recombination event
between marker 3
and gl1
Is gl1 closer to the
centromere or the
telomere?
Marker 5 is centromeric
Marker 4 is telomeric
Plants 3, 4, 5 and 10
are useful to identify
flanking markers
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 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
C/C at 5
Col-0
La-er
Plant 5
Plant 3
5
5
5
5
5
5
gl1
gl1
gl1
gl1
gl1
gl1
3
3
3
3
3
3
4
4
4
4
4
4
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.
We will analyze the alleles of new markers
located between marker 3 and marker 5.
We will only analyze DNA from plants heterozygous
at either marker 3 or marker 5.
From comparison of genome sequence to a
recombination map made by Lister and Dean,
we learned that Arabidopsis has approximately
250 kb per map unit. That represents about 100 genes.
For convenience, we aim for map resolution of 0.1
map units, which should represent 25-100 kb and
hopefully 10-20 genes.
50-100 kb is the normal insert size for BAC clones.
In order to get to map resolution of 0.1 map units we
screen at least 1000 F2 plants (2000 chromosomes)
How to decide the number of F2’s to examine?
Recombination frequency is calculated:
Number of recombinants/number of chromosomes,
1 recombinant chromosome/2000 chromosomes
= 0.05 map units.
We can only find a locus by identification of recombination events
on either side.
Therefore, with 2000 chromosomes we should find one marker
0.05 cM to the right of GL1 and another marker 0.05 cm to the left.
An interval of 0.1 map units between the two closest markers
is the best we measure.
If we want better resolution, we need more markers (which we have)
and more potential recombinant chromosomes from F2 plants.
Once we have defined 2 markers flanking our interval that are physically
close enough, we start sequence analysis for point mutations.
MDF20
MYN21
BAC T22A15 100 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.
Go to TAIR for GL1 marker on AGI map click clone, then look at gene annotation
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.
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.
Grant et al 1995 Science 269;843-846.
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.
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.