A series of experiments narrows the mystery down to one candidate gene.
You might not think about it when you prepare eggs for breakfast, but the color of an egg is an important ecological trait. The eggshell color is mainly determined by a mixture of three types of pigments: protoporphyrin-IX, biliverdin-IX and biliverdin zinc chelate. These pigments provide protection against damaging solar radiation and play a role in thermoregulation, creating the ideal conditions for embryonic development. Moreover, egg color is often a crucial factor in brood parasites that mimic the egg color of their unsuspecting hosts. In some species, the color of eggs is even used as a signal for female quality (see for example this blog post on hoopoes).
Despite this variety of roles in ecological and evolutionary processes, the genetic basis of egg color remains largely unknown. A recent study in the journal Molecular Ecology focused on the green egg color of a Chinese domesticated duck breed (the Jinding duck). What genes underlie this peculiar green color?
The researchers performed a series of clever experiments to determine the genetic basis of green egg color (similar to the case of the mosaic canary described in this blog post). First, they compared the genomes of reciprocal crosses between Pekin ducks (with white eggs) and mallards (with green eggs). The genome-wide search identified a broad target region on chromosome 4 that was significantly associated with egg color. This region was explored in greater detail by analyzing the genomes of seven indigenous duck populations that differ in the coloration of their eggshells. The divergence between green-shelled and white-shelled populations could be traced to a small section of the target region, containing two genes: PRKG2 and ABCG2. The second gene (ABCG2) codes for a membrane transporter that carries biliverdin, one of the pigments that contribute to egg shell coloration. Sounds like the perfect candidate gene.
The researchers did not stop at identifying the candidate gene. They performed more detailed analyses to understand how this gene contributes to the green egg color. Eggshell formation takes place in the uterus, so the researchers measured gene expression in uterine tissues from four populations. As expected, ABCG2 was expressed at a higher level in the green-shelled groups compared to the white-shelled groups. Moreover, the data revealed that ABCG2 produces five distinct isoforms (i.e. different proteins that derive from the same DNA sequence). This is achieved by combining different sections of the gene during protein translation. In the mallard case, the third isoform (ABCG2-X3) is expressed most.
Finally, the researchers took a closer look at the DNA sequence of ABCG2. Does it contain genetic differences that clearly separate green-shelled from white-shelled ducks? Using a collection of sophisticated analyses (including ATAC-sequencing and a luciferase assay), the search could be narrowed down to one genetic variant on nucleotide 47,418,074 of chromosome 4. This position is targeted by different transcription factors (ATF and c/EBPα) in white-shelled compared to green-shelled ducks: ATF binds the white-shelled variant, whereas c/EBPα binds the green-shelled variant. The green egg color of the Jinding duck can thus be explained by a regulatory change in the expression of a specific isoform of the ABCG2-gene. The resulting protein is active in the uterus where it transports the pigment biliverdin from the blood onto the developing egg shell. Mystery solved.
Liu et al. (2021). A single nucleotide polymorphism variant located in the cis‐regulatory region of the ABCG2 gene is associated with mallard egg colour. Molecular Ecology, 30(6), 1477-1491.
Featured image: Green eggs in the nest of a duck © Smudge9000 | Flickr