Figure 3

RAD mapping using bulk segregant analysis. (a) F₁ fish heterozygous for the mutation and a polymorphic mapping strain are crossed together. (b) The F₂, a few of which are shown here, will contain a large variety of recombinant chromosomes. Pools of DNAs from either mutant or phenotypically wild-type animals are made. Marker RAD2, for example, is closely linked to the wild-type allele of the mutant locus, and therefore (except for rare recombinants) will appear in the wild-type pool but not in the mutant pool. (c) Restriction site associated DNA (RAD) tags are isolated from each of the pools, fluorescently labeled, and hybridized to the RAD marker microarray. Markers unlinked to the mutant locus should be in approximately equal quantities in both pools. As markers become closer to the mutation, the fraction of individuals that have one or the other RAD marker allele will increase, resulting in array elements with high or low ratios of red to green fluorescence.