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.

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 interestthat define an interval on a chromosome.

Markers that define major regions of the Arabidopsis chromosomes

F2s are selected as homozygous recessivegl1/gl1 by phenotype

eg. Scored for 5 markers

1, 2 are not linked to GL1 13: 25: 12C/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 LandsbergThe 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 cM3 is 4/100 X100 cM from GL1 = 4 cm 4 is 30 cM5 is 20 cM

Markers 3, 4 and 5 are linked to Gl1

Need to find another markeron the opposite side to definethe interval that contains GL1.

gl1

3

gl1

3

Markers 3, and 5 flank GL1

gl1

3

There has been no crossover between GL1 and 5And 5 is further away from GL1 than 3.

This means 3 and 5 define the interval that contains GL1.

Col-0

La-er 4

5

Plant 3

Plant 3 is C/L at 3L/L at 4 andC/C at 5

Plants 3, 4, 5, 8 and 10 are useful to find closer markers

There is a recombination event either between 3 and gl1or 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 3 is C/L at 3L/L at 4 andC/C at 5

Plant 4 is C/C at 3C/C at 4 andC/Lat 5

Plant 5 is C/L at 3C/L at 4 andC/C at 5

Col-0

La-er

gl1

3

4

5

Plant 4

Plant 3

Plant 5

gl1

3

4

5

gl1

3

4

5

gl1

3

4

5

gl1

3

4

5

gl1

3

4

5

Using closely linked Markers to identify asmall interval containing GL

Once we find flanking markersthat 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

MDF20 MYN21

BAC T22A15 100 kb insert

BAC sequence gives us a list of genes.Candidate genes can be PCR amplified from the mutantand 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 orTransform mutant plant with wild type gene for complementation.

Once we have defined 2 markers flanking our interval that are physically close enough, we start sequence analysis for point mutations.

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.