Regulatory and structural variants in the CLCN2 gene are associated with modified skin colour pattern phenotypes in the corn snake

Background: Snakes exhibit a broad variety of adaptive colours and colour patterns, generated by the spatial arrangement of chromatophores, but little is known of the mechanisms responsible for these spectacular traits. Here, we investigate a monolocus trait with two recessive alleles that cause pattern aberrations in the corn snake: the motley and striped alleles. Results: We use mapping-by-sequencing to identify the genomic interval where the causal mutations reside. With our differential gene expression analyses, we find that CLCN2 (Chloride Voltage-Gated Channel 2), a gene within the genomic interval, is significantly downregulated in Motley embryonic skin. Furthermore, the insertion of a retrotransposon in CLCN2 results in a disruptive mutation in Stripe. We confirm the involvement of CLCN2 in colour pattern formation by producing knock-out snakes that present a phenotype similar to the Stripe one. In humans and mice, the disruption of CLCN2 results in leukoencephalopathy, as well as retinal and testes degeneration. Our single-cell transcriptomic analyses reveal that CLCN2 is indeed expressed in chromatophores during embryogenesis and in the adult brain. Although, we observe signs of vacuolation in the Stripe adult brain, the retina of Motley and Stripe animals is intact, and no fertility issues are reported for the carriers of both alleles. Conclusions: Our genomic, transcriptomic and functional analyses identify a plasma membrane anion channel to be involved in colour pattern development in Squamate reptiles and show that an active LTR-retrotransposon might be a key driver of trait diversification in corn snakes. We performed single-nuclei RNAseq on brain and spinal cord from an adult Stripe corn snake using the 10x Genomics Chromium 3'GeneExpression kit. The cDNA libraries were quality controlled using a 2100 Bioanalyzer and TapeStation from Agilent and sequenced using a HiSeq 2500 sequencer from the iGE3 platform at the University of Geneva