The role of cytogenetics in the chromosome-level genomic era

Abstract Recent advances in long-read sequencing and proximity-ligation technologies have enabled the routine generation of “chromosome-level” genome assemblies across diverse taxa. However, equating assembled scaffolds with true biological chromosomes remains a widespread misconception. Chromosomes are physical, hierarchical structures whose organization and behavior cannot be fully inferred from sequence data alone. As a result, important evolutionary events, including fusions, fissions, inversions, and translocations, may be overlooked or misinterpreted when genome assemblies lack cytogenetic validation. In this review, we provide a timely synthesis of the historical and continuing role of cytogenetics in genome research and assess how modern cytogenomic approaches, particularly oligonucleotide-based fluorescence in situ hybridization (Oligo-FISH), can be integrated into contemporary assembly workflows. We argue that the marginalization of cytogenetics in the genomics era has created a critical gap between in silico assemblies and native chromosomal architecture. Our contribution is a conceptual framework and a practical, integrative pipeline that positions cytogenetic evidence as an essential component of genome projects, rather than a secondary validation step. By bridging genomics and chromosome biology, this review highlights how incorporating cytogenetic data can improve the accuracy, interpretation, and evolutionary relevance of chromosome-scale assemblies, with implications for biodiversity research, agriculture, and biomedicine.