A large deletion at the cortex locus eliminates butterfly wing patterning

Abstract

As the genetic basis of natural and domesticated variation has been described in recent years, a number of hotspot genes have been repeatedly identified as the targets of selection, Heliconius butterflies display a spectacular diversity of pattern variants in the wild and the genetic basis of these patterns has been well-described. Here we sought to identify the mechanism behind an unusual pattern variant that is instead found in captivity, the ivory mutant, in which all scales on both the wings and body become white or yellow. Using a combination of autozygosity mapping and coverage analysis from 37 captive individuals, we identify a 78kb deletion at the cortex wing patterning locus, a gene which has been associated with wing pattern evolution in H. melpomene and 10 divergent lepidopteran species. This deletion is undetected among 458 wild Heliconius genomes samples, and its dosage explains both homozygous and heterozygous ivory phenotypes found in captivity. The deletion spans a large 5’ region of the cortex gene that includes a facultative 5’UTR exon detected in larval wing disk transcriptomes. CRISPR mutagenesis of this exon replicates the wing phenotypes from coding knock-outs of cortex, consistent with a functional role of ivory-deleted elements in establishing scale color fate. Population demographics reveal that the stock giving rise to the ivory mutant has a mixed origin from across the wild range of H. melpomene, and supports a scenario where the ivory mutation occurred after the introduction of cortex haplotypes from Ecuador. Homozygotes for the ivory deletion are inviable while heterozygotes are the targets of artificial selection, joining 40 other examples of allelic variants that provide heterozygous advantage in animal populations under artificial selection by fanciers and breeders. Finally, our results highlight the promise of autozygosity and association mapping for identifying the genetic basis of aberrant mutations in captive insect populations.

TheivorylncRNA regulates seasonal color patterns in buckeye butterflies

“…3C). This phenotype resembled the original H. melpomene ivory spontaneous mutants (13), where almost all melanins appeared to be lost, and only weak traces of presumptive ommochrome pigmentation remained. ivory Δ17kb did not prove to be embryonic lethal, although homozygous butterflies were flightless and not as active as heterozygotes or wildtypes.…”

“…These data identified a lncRNA that partially overlaps with the 5' end of the cortex gene. The 3' region of this predicted lncRNA also overlaps with the previously described 78kb ivory mutation -a spontaneous deletion identified in Heliconius melpomene that produced largely unpigmented butterflies (13) -thus, this novel lncRNA is dubbed ivory after the original Heliconius mutation.…”

“…Second, deletions at all elements were all capable of producing strongly hypomorphic mutant clones that resembled the ivory ∆17kb line, and as evidenced in other species (6,13) -an almost complete loss of most pigmentation on both dorsal and ventral wing surfaces (Fig. 4D).…”

A micro-RNA is the effector gene of a classic evolutionary hotspot locus

“…When the effector gene is a poorly annotated non-coding element that acts in trans , such as mir-193 or its primary transcript ivory investigated in this study, a direct link between local genetic variation and flanking protein-coding genes can lead to misleading conclusions. This happened with the cortex locus where a large number of intraspecific wing color pattern polymorphisms in lepidopterans, in most cases involving black and white, or dark and bright color patterns, have been mapped to this hotspot locus and been mistakenly attributed to cortex regulation ( 2, 5, 7, 15, 17 ). It will be interesting to examine in future how the genomic contexts and targets of mir-193 have evolved across animals and how, ebony , one of the targets of mir-193 in B. anynana , became the hotspot locus across Drosophila species ( 37 ), despite the presence and conserved function of mir-193 .…”

“…Functional studies using CRISPR-Cas9 suggested that cortex might promote wing melanization. These studies, however, suffered from low success rates ( 2, 15, 17 ), hinting that hidden genomic features next to cortex , rather than cortex itself, could be causative. While previous investigations exclusively focused on protein-coding genes, non-coding RNAs were ignored, despite the presence of two micro-RNAs (miRNAs) at this locus ( 18 ).…”