Mapping in Arabidopsis cont… Jeff Long Salk Institute

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

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

SSLP’s can be tracked by PCR with no digestion

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

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

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.

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

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.

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

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

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

Mapping primer positions 1 2 3