The complete genome of Trypanosoma cruzi reveals 32 chromosomes and three genomic compartments

Trypanosoma cruzi, the causative agent of Chagas disease, exhibits remarkable genomic variability and possesses an expanded genome rich in multigene families. However, its precise chromosomal composition has remained elusive due to the challenges posed by extensive repetitive regions. In this work, we determined the complete molecular karyotype of the T. cruzi Dm28c strain, which comprises 32 chromosomes. Comparison with two independently isolated Clade A (TcI) strains revealed clear chromosomal homology, indicating that the 32-chromosome karyotype is stable, at least among this clade. T. cruzi is diploid for all chromosomes, except chromosome 16, which is consistently tetrasomic in all strains analyzed. This tetrasomic chromosome is homologous to chromosome 31 of Leishmania major, which is also tetrasomic. Through a comprehensive annotation pipeline, we refined gene content and resolved haplotypes. Comparative analysis of chromosomal architecture revealed that all chromosomes exhibit a conserved distribution of core and disruptive compartments, along with notable conservation in GC content transitions. These findings demonstrate a high degree of structural conservation, challenging the prevailing paradigm of extensive chromosomal rearrangements and high genomic plasticity in T. cruzi. Subtelomeres—enriched in RHS, DGF-1, and TS genes, and depleted in MASP and mucins—constitute a distinct third genomic compartment that is transcriptionally active and represents the primary source of interstrain genomic variability. Furthermore, a reclassification of the TS gene family revealed differential distribution patterns across subtelomeric and disruptive compartments. Altogether, this work defines the complete chromosomal complement of T. cruzi, establishing a robust framework for comparative genomics and enabling detailed investigations into genome organization, antigenic variability, and evolutionary dynamics across strains and clades, opening new avenues for exploring T. cruzi biology, pathogenic diversity, and adaptive mechanisms.