The Bovidae CSN2 Locus as a Multilayered Evolutionary System: Evidence from Retroposons, Trans-Species Variation and Ancient Recombination

The β-casein gene (CSN2) encodes one of the major milk proteins involved in calcium and phosphorus transport and combines a highly conserved genomic organization with extensive genetic diversity, making it an informative model for investigating mammalian genome evolution. Although numerous studies have described CSN2 polymorphisms, particularly in domestic species, the broader evolutionary history of the locus remains poorly understood. Here, we investigated the evolutionary architecture of CSN2 across Bovidae and related Cetartiodactyla by integrating coding variation, non-coding polymorphisms, retroposon insertions and recombination analyses. Comparative genomic analyses identified previously undescribed caprine haplotypes, expanded the known spectrum of β-casein variation in domestic and wild Caprinae, and revealed multiple structural configurations of the locus, including distinct retroposon architectures and intronic insertion/deletion polymorphisms. Retroposon mapping and comparative analyses supported a hierarchical accumulation of structural variants, while also indicating ancestral polymorphism and lineage-specific retention in rapidly radiating caprine lineages. A locus-level phylogenetic analysis revealed two deeply divergent allelic lineages and provided evidence of ancient interallelic recombination predating the divergence of Capra and Ovis. Comparative analyses further showed that a two-amino-acid deletion previously associated mainly with Caprinae is more widely distributed across Bovidae than previously recognized. Overall, the CSN2 locus emerges as a multilayered evolutionary system shaped by structural variation, ancestral polymorphism, incomplete lineage sorting and ancient recombination, illustrating how the integration of coding, structural and retroposon variation can improve locus-level evolutionary reconstruction in Bovidae.