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Evidence of whole-genome duplications (WGDs) and subsequent karyotype changes has been detected in most major lineages of living organisms on Earth. To clarify the complex resulting multi-layered patterns of gene collinearity in genome analyses, there is a need for convenient and accurate toolkits. To meet this need, we developed WGDI (Whole-Genome Duplication Integrated analysis), a Python-based command-line tool that facilitates comprehensive analysis of recursive polyploidization events and cross-species genome alignments. WGDI supports three main workflows (polyploid inference, hierarchical inference of genomic homology, and ancestral chromosome karyotyping) that can improve the detection of WGD and characterization of WGD-related events based on high-quality chromosome-level genomes. Significantly, it can extract complete synteny blocks and facilitate reconstruction of detailed …