The study supports the idea that an ancient color pattern-based program is already encoded in butterfly genomes, with noncoding regulatory DNA working like switches to turn some patterns on and turn off others.
Gulf fluffy butterfly — Agraulis vanillae. Source: Anyi Mazo-Vargas
“We’re curious to see how the same gene builds these very different-looking butterflies,” said the study’s lead author and senior author Robert Reid, a former graduate student in the lab of Ecology and Evolution in the College of Agriculture and Life Sciences Biology professor Anyi Mazo-Vargas, 20, Ph.D. Mazo-Vargas is currently a postdoctoral fellow at George Washington University.
“We saw that there is a very conserved set of switches [non-coding DNA] that work at different locations and are activated and drive genes,” Mazo-Vargas said.
Previous work in Reed’s lab has uncovered key color-patterning genes: one (WntA) controls stripes and another (Optix) controls butterfly wing color and iridescence. When the researchers disabled the Optix gene, the wings appeared black, while when the WntA gene was deleted, the striped pattern disappeared.
Detail of the wing pattern of the Gulf velvet butterfly (Agraulis vanilla), which is altered by modifying noncoding DNA sequences using the gene editing tool CRISPR/cas9. Source: Anyi Mazo-Vargas
This study focused on the effect of noncoding DNA on the WntA gene. Specifically, the researchers conducted experiments with 46 of these noncoding elements in five species of Nymphalidae, the largest family of butterflies.
In order for these noncoding regulatory elements to control the gene, the tightly wound coils of DNA are unwound, a sign that the regulatory element interacts with the gene to turn it on, or in some cases, turn it off.
A mutant monarch butterfly (Danaus plexippus) that uses the gene-editing tool CRISPR/cas9 to delete a noncoding DNA sequence, also known as “junk DNA,” that regulates a gene that controls wing shape. Source: Anyi Mazo-Vargas
In the study, the researchers used a technique called ATAC-seq to identify the regions in the genome where this unraveling is taking place. Mazo-Vargas compared ATAC-seq maps from five species of butterfly wings to identify genetic regions involved in airfoil development. They were surprised to find that a large number of regulatory regions are shared among very different butterfly species.
Mazo-Vargas and colleagues then used CRISPR-Cas gene editing technology to disable 46 regulatory elements at a time to see how these noncoding DNA sequences were disrupted on wing shape. When removed, each non-coding element changes one aspect of the butterfly’s wing pattern.
The researchers found that in four species — Junonia coenia (buckeye), Vanessa cardui (painted lady), Heliconius himera and Agraulis vanillae (gulf fritillary) — these noncoding elements function similarly to the WntA gene, proving that they are ancient and conserved, possibly originating from a distant common ancestor.
They also found that D. plexippus uses different regulatory elements to control its WntA gene than the other four species, perhaps because it has historically lost some genetic information and had to reinvent its own regulatory system to develop its unique coloration model.
“We’ve come to understand that most evolution occurs due to mutations in these noncoding regions,” Reed said. “My hope is that this paper will be a case study of how people can use this combination of ATAC-seq and CRISPR to start interrogating these interesting regions in their own research systems, whether they are in birds, Flies still work on worms.”
“This study is a breakthrough in our understanding of the genetic control of complex traits, and not just in butterfly populations,” said Theodore Morgan, a program director at the National Science Foundation. “This study not only shows how the instructions for butterfly color patterns are deeply conserved throughout evolutionary history, but also reveals new evidence of how regulatory DNA segments can positively and negatively influence traits such as color and shape.”