The evolution of <i>Sex-linked barring</i> alleles in chickens involves both regulatory and coding changes in <i>CDKN2A</i>

<div><p>Sex-linked barring is a fascinating plumage pattern in chickens recently shown to be associated with two non-coding and two missense mutations affecting the ARF transcript at the <i>CDKN2A</i> tumor suppressor locus. It however remained a mystery whether all four mutations are indeed causative and how they contribute to the barring phenotype. Here, we show that Sex-linked barring is genetically heterogeneous, and that the mutations form three functionally different variant alleles. The <i>B0</i> allele carries only the two non-coding changes and is associated with the most dilute barring pattern, whereas the <i>B1</i> and <i>B2</i> alleles carry both the two non-coding changes and one each of the two missense mutations causing the Sex-linked barring and Sex-linked dilution phenotypes, respectively. The data are consistent with evolution of alleles where the non-coding changes occurred first followed by the two missense mutations that resulted in a phenotype more appealing to humans. We show that one or both of the non-coding changes are <i>cis</i>-regulatory mutations causing a higher <i>CDKN2A</i> expression, whereas the missense mutations reduce the ability of ARF to interact with MDM2. Caspase assays for all genotypes revealed no apoptotic events and our results are consistent with a recent study indicating that the loss of melanocyte progenitors in Sex-linked barring in chicken is caused by premature differentiation and not apoptosis. Our results show that <i>CDKN2A</i> is a major locus driving the differentiation of avian melanocytes in a temporal and spatial manner.</p>