Satellite DNA landscape in the alpaca genome and comparison with other camelids

Abstract Satellite DNAs (satDNAs) are among the least resolved components of telomere-to-telomere (T2T) genome assemblies despite recent advances in long-read sequencing. Among mammals, camelids provide an excellent system to study satDNA evolution because of their exceptional karyotypic conservation despite more than 11 million years of independent evolution. Here, we systematically characterize the satDNA landscape of the chromosome-level alpaca reference genome VicPac4 by combining bioinformatic reference-guided repeat identification, PCR validation, and fluorescence in situ hybridization (FISH). Cross-species PCR analysis of 13 candidate satDNAs revealed two major conservation patterns: six families amplified across all camelids, whereas seven were restricted to South American camelids (SACs). FISH further demonstrated distinct chromosomal organization of nine satDNA families: four showed widespread chromosomal distribution; three localized near centromeres of a subset of chromosomes; one was primarily associated with chromosome 26 but had lower copy number on additional chromosomes, and one exclusively mapped to chromosomes 27 and 36 in all SACs. The latter displayed consistent and prominent copy number differences between species, individuals and homologous chromosomes, suggesting naturally occurring variation in satellite array expansion. This satellite amplified by PCR across Camelidae, whereas FISH detected expanded arrays only in SACs, suggesting differences in satellite organization between SAC and Camels. Beyond satDNA identification, the proposed workflow facilitated the discovery and comparative analysis of pseudogene-rich repetitive blocks in the alpaca genome and their absence in Camels’ genome assemblies. These findings substantially expand the alpaca satDNAs catalogue and provide a cytogenetic framework for future T2T genome refinement and comparative studies across Camelidae.