Tobacco karyotyping by accurate centromere identification and novel repetitive DNA localization

Tobacco ( Nicotiana tabacum ) is an amphidiploid species (2 n = 4 x = 48, genome constitution SSTT) derived from a natural hybrid between Nicotiana sylvestris (2 n = 2 x = 24, SS) and Nicotiana tomentosiformis (2 n = 2 x = 24, TT). Genomic in situ hybridization (GISH), using the genomic DNA from these ancestral species as probes, revealed the chromosomal origins (S or T) and the occurrence of intergenomic translocations in N . tabacum . Fluorescence in situ hybridization (FISH) was also used to distinguish between chromosomes. However, the use of repetitive DNA sequences as probes for FISH analysis is limited by an inability to identify all chromosomes. In addition to this limitation, the occurrence of chromosomal tertiary constrictions can easily lead to the misclassification of chromosomes. To overcome these issues, immunostaining with anti- N . tabacum centromere-specific histone H3 antibody was carried out to determine the centromere position of each chromosome, followed by FISH analysis with ten distinct repetitive DNA probes. This approach allowed us to identify 22 of the 24 chromosome pairs in N . tabacum and revealed novel intergenomic chromosome rearrangements and B-chromosome-like minichromosomes. Hence, the combination of immunostaining with FISH and GISH is critical to accurately karyotype tobacco.