A 16-bp deletion in the first intron of Eda is not responsible for the loss of lateral plates in threespine stickleback

While much progress has been made in identifying the genes and genomic regions underlying adaptive phenotypic evolution, identifying the causal mutations remains challenging. For example, the gene Ectodysplasin A (Eda) mediates the loss of lateral plates in freshwater populations of the threespine stickleback (Gasterosteus aculeatus Linnaeus, 1758). However, the causative mutation underlying the phenotypic effects of Eda is unknown, in part due to strong linkage disequilibrium in the 16 kb haplotype containing Eda. An association mapping study in a population with historical recombination within this 16 kb haplotype previously demonstrated that the causative mutation is in a 1.4 kb region in the first intron of Eda. Within this intronic region, we identified a 16 bp deletion (LP3621) in the freshwater haplotype as a candidate causative mutation. We generated a variety of mutations in LP3621 on the marine haplotype of heterozygous fish using CRISPR-Cas9. However, even complete deletions of LP3621 did not recapitulate the loss of lateral plates. These results suggest that the causative mutation is either somewhere else within the 1.4 kb region or that LP3621 interacts with another mutation in the Eda haplotype to cause the loss of lateral plates in threespine stickleback.