Download all chromosomes recombine in meiosis F2 plants

Finding a gene based on phenotype
Model organisms
• 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.
Parents: Col-0 a/a 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?
Mouse mapping Panels
Backcross panel
Cross parents from inbred lines, B and S
BXS heterozygote cross to either B or S
Progeny are recombinant BS/parental S
– like a test cross.
Each mouse represents one BS recombinant
chromosome.
Parents: Col-0 a/a X La-er GL1/GL1 genotypes
F1 is self fertilized –
all chromosomes recombine in meiosis
F2 plants –
recombined chromosomes segregate
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.
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
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
Marker 1 from a previous year.
The first lane is the glabrous mutant (Columbia),
the second lane is a mixture of 1 and 3
The third lane is Landsberg
The rest are DNA from F2 plants
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
Markers 3, 4 and 5 are linked to Gl1
Need to find another marker
on the opposite side to define
the interval that contains GL1.
gl1
gl1
3
3
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
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
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
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.
Making a library of clones that represent the entire genome Putting them in order to represent the genome sequence
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
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.
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.
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.