Finding a gene based on phenotype model organisms
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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

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Finding a gene based on phenotype Model organisms

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Finding a gene based on phenotype model organisms

Finding a gene based on phenotypeModel 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.


Finding a gene based on phenotype model organisms

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

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.


Finding a gene based on phenotype model organisms

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

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.


Finding a gene based on phenotype model organisms

Interval mapping:

Identify markers linked to the gene of interest

that define an interval on a chromosome.


Finding a gene based on phenotype model organisms

Markers that define major regions of the

Arabidopsis chromosomes


Finding a gene based on phenotype model organisms

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


Finding a gene based on phenotype model organisms

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


Finding a gene based on phenotype model organisms

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


Finding a gene based on phenotype model organisms

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


Finding a gene based on phenotype model organisms

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

4

La-er


Finding a gene based on phenotype model organisms

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


Finding a gene based on phenotype model organisms

5

5

5

5

5

5

gl1

gl1

gl1

gl1

gl1

gl1

3

3

3

3

3

3

4

4

4

4

4

4

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

C/C at 5

Plant 3

Col-0

La-er


Finding a gene based on phenotype model organisms

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.


Finding a gene based on phenotype model organisms

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

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


Finding a gene based on phenotype model organisms

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

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

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.


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