Fig. 6.

Multi-allelic phenotyping – round 2. Round 2 pairs are set up based on the genotyping results of the first round phenotyping. Clutches are collected and observed over the first five days of development. (a) When phenotypes arise in approximately 25% of embryos, they are collected along with non-phenotypic siblings and frozen in MeOH. Multiple phenotypes per clutch can be collected simultaneously but are archived in separate tubes. Phenotypic embryos are then aliquoted into the upper half of a 96 well plate. (b) Multiple phenotypes can be added to a single plate and multiple plates per clutch can be produced. Phenotypically normal siblings are then added to the lower half of the 96 well plates with wells H11 and H12 containing parental control DNA. The 96 well plates are then transferred into 384 well plates where all alleles flagged for each particular pair combination are assayed against all identified phenotypes per clutch. (c) A KASP Kluster Caller plot depicting the grouping of the three genotypes. (d) This is an example of a genotype-phenotype association that would be documented as likely causal (e).