Revisiting the cytogenetics of Vellozia Vand.: immunolocalization of KLN1 elucidates the chromosome number for the genus

Chromosome number is the most fundamental trait of a karyotype. Accurate chromosome counting is essential for further analyses including cytogenomics, taxonomic, evolutionary, and genomic studies. Despite its importance, miscounting is common, especially in early publications on species with small and morphologically similar chromosomes. Vellozia Vand. is a genus mainly distributed throughout South America belonging to the angiosperm family Velloziaceae, a dominant taxon in the Brazilian "campos rupestres". Cytogenetic studies within the group have been rare and have shown conflicting chromosome counts, even within the same species. These discrepancies are associated with the presence of a few small chromosome-like structures, which were previously classified as possible satellites. Here, to accurately determine the chromosome number of species belonging to the genus, we used different cytogenomics approaches, including the immunostaining of the KNL1 kinetochore protein combined with chromosome spread preparation using tissue culture-derived samples. Our results revealed 2n = 18 chromosomes for all six species studied. This finding suggests that the basic chromosome number for Vellozia is x = 9 and not x = 8, as previously proposed. The immunolocalization of functional centromeres was fundamental for undoubtedly identifying the smaller chromosome pair as real chromosomes and accurately determining the correct chromosome number of these species. This will provide substantial support for further studies, including investigations into karyotype evolution and the generation of reference genomes for the species of the family.