1. Mapping in Arabidopsis cont…
Jeff Long
Salk Institute

2. 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

3. F1 plant
F2 gametes

4. Can use DNA changes between ecotypes
as markers for mapping
The example below is a simple sequence length
polymorphism-SSLP’s
Ler
Col

5. SSLP’s can be tracked by PCR with
no digestion

6. F2 chromosomes
Mutant X different ecotype
tpl-1
Self F1 plants
marker 1
Select mutant F2 plants
for mapping
marker 2

7. By picking homozygous mutants, you
know the ecotype at the mutant locus
Markers close to your gene will show
a bias toward the original ecotype
Referred to as linkage
tpl-1
marker 1
marker 2
F2 chromosomes
Ler
Col
9/10 Ler
5/12 Ler
Ler
Col

8. How does recombination frequency get you to
your gene?
tpl-1
marker 1
marker 2
F2 chromosomes
Ler
Col
9/10 Ler
With marker 1, we have 1 recombinant chromosome out of 10. Therefore we have 10% recombination frequency.

9. Recombination frequency can give you a rough estimate of
distance.
1%recombination=1Centimorgan (Cm) or map unit
In Arabidopsis, 1Cm= 150 Kbp (roughly)

10. 9/10 Ler tpl-1 marker 1 marker 2 F2 chromosomes 10 Cm
Col
9/10 Ler
tpl-1
marker 1
marker 2
F2 chromosomes
10 Cm
If tpl-1 shows 10% recombination between it and marker 1, we know that tpl-1 is roughly 1500 Kbp away from
This is likely wrong-need many more chromosomes (like 1000) to get a good map distance.

11. Once you have narrowed down your gene location
you can design new primers in the interval
Known
Markers
BAC
clones

12. Databases such as TAIR mapviewer can tell you what
genes are in your interval

13. Once you have narrowed down your mapping region
to <30 genes, you can take a candidate gene approach
Sequence genes in region and compare to wild-type
sequence in publicly available database (TAIR, NCBI)
If mutation is found, have to confirm it is the right gene
-sequence a second allele if available
-order an insertion line and compare the phenotype
-attempt to rescue the mutant with a wild-type version of
the candidate gene

14. Mapping primer positions1 2 3