A novel CENH3-based immunostaining method for ploidy estimation in potato

Accurate and reliable ploidy estimation is essential for potato genetic improvement programmes, yet current methods face limitations in cost, throughput, or technical complexity. Here, an immunofluorescence assay was developed that employs a species-specific antibody to label the centromeric histone CENH3 in Solanum leaf cell nuclei. The method was validated on genotypes of known ploidy (2x, 3x, 4x) and compared against flow cytometry and root-tip chromosome counting. The antibody specificity was examined across a range of both related and distant plant species. The results demonstrated that CENH3 signals were both clear and quantifiable, with mean counts per nucleus (21.4, 31.7, and 42.1 for 2x, 3x, and 4x, respectively) demonstrating robust distinction among ploidy levels (P < 0.001). Ploidy calls were consistent with the results of the flow cytometry and root-tip chromosome counting assays. Furthermore, the method enabled the identification of mixoploid cells. Notably, the antibody was effective across different Solanum species (both wild and cultivated) and across different leaf sampling positions, demonstrating its broad applicability and sampling flexibility. The antibody produced specific signals exclusively in Solanum, exhibiting no cross-reactivity in related Solanaceae species or distantly related species. The CENH3 immunofluorescence method developed in this study offers an accurate, reliable and instrument-flexible alternative for potato ploidy screening and breeding.