Presentation on theme: "Mapping in Arabidopsis cont…"— Presentation transcript:
1Mapping in Arabidopsis cont… Jeff LongSalk Institute
2Mapping strategy Cross your mutant to a different ecotype (our case mutant is in Landsberg erecta and different ecotype isColumbia)Allow the F1 population to self after meiosis (whererecombination will take place)Isolate DNA from resulting F2 mutant plants- use DNAdifferences between ecotypes to map
6F2 chromosomesMutant X different ecotypetpl-1Self F1 plantsmarker 1Select mutant F2 plantsfor mappingmarker 2
7By picking homozygous mutants, you know the ecotype at the mutant locusMarkers close to your gene will showa bias toward the original ecotypeReferred to as linkagetpl-1marker 1marker 2F2 chromosomesLerCol9/10 Ler5/12 LerLerCol
8How does recombination frequency get you to your gene?tpl-1marker 1marker 2F2 chromosomesLerCol9/10 LerWith marker 1, we have 1 recombinant chromosome out of 10. Therefore we have 10% recombination frequency.
9Recombination frequency can give you a rough estimate of distance.1%recombination=1Centimorgan (Cm) or map unitIn Arabidopsis, 1Cm= 150 Kbp (roughly)
109/10 Ler tpl-1 marker 1 marker 2 F2 chromosomes 10 Cm Col9/10 Lertpl-1marker 1marker 2F2 chromosomes10 CmIf tpl-1 shows 10% recombination between it and marker 1, we know that tpl-1 is roughly 1500 Kbp away fromThis is likely wrong-need many more chromosomes (like 1000) to get a good map distance.
11Once you have narrowed down your gene location you can design new primers in the intervalKnownMarkersBACclones
12Databases such as TAIR mapviewer can tell you what genes are in your interval
13Once you have narrowed down your mapping region to <30 genes, you can take a candidate gene approachSequence genes in region and compare to wild-typesequence 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 ofthe candidate gene