Mapping in Arabidopsis

Jeff Long Salk Institute

Cotyledons (seed leaves)Shoot Apical Meristem

Hypocotyl (seedling stem)

Root

Root Apical Meristem

WT

tpl-1 mutant phenotypes

monocot

tpl-1

tube

tpl-1

pin

tpl-1

One way to learn about development is by examining mutants

One way to learn about development is by examining mutants

How does one generate mutants?

T-DNA mutagenesis

Mutagens that create base pair changes

EMS, Gamma Rays, Others

Problem-How do you identify the mutated gene from the other 25,000 found in the genome?

The answer is by mapping!

In Arabidopsis we can take advantage of DNA differences between different strainsor ecotypes.

What is an ecotype?

An ecotype is just another strain of Arabidopsisthat has been geographically isolated

Different ecotypes often contain differences in their DNA

These differences can destroy or create newrestriction sites

Known as restriction fragment length polymorphismsor RFLP’s

These RFLP’s can be tracked either by Southern blotting or PCR followed by digestion

promoter coding

Sequence differences can be found between ecotypes

CCAAGCTGAATTCTTCGAAT Col

CCAAGCTGTATTCTTCGAAT Ler

EcoR1

Ler Col

Ler Col

Ler Col

Ler

Col

DNA changes can also cause insertions/deletions-Indels

Can use PCR primers to track these changes=markers

The example below is a simple sequence length polymorphism-SSLP’s

Indels can be tracked by PCR with no digestion

Mapping strategy

Cross your mutant to a different ecotype (our case mutant is in Landsberg erecta and different ecotype is Columbia)

Allow the F1 population to self after meiosis (where recombination will take place)

Isolate DNA from resulting F2 mutant plants- use DNA differences between ecotypes to map

F1 plant

F2 gametes

Mutant X different ecotype

Self F1 plants

Select mutant F2 plantsfor mapping

mutant

marker 1

marker 2

F2 chromosomes

mutant

marker 1

marker 2

Markers close to your gene will showa bias toward the original ecotypeReferred to as linkage

LerCol9/10 Ler

F2 chromosomesBy picking homozygous mutants, you know the ecotype at the mutant locus

5/12 LerLerCol

monocot

tpl-1

Isolate DNA from tpl-1 mapping population