Determining relative abundance of specific DNA sequences in flow cytometrically sorted maize nuclei

A wide breadth of DNA content variation has been reported among maize lines. While the extent of this variation has been well documented, few studies have focused on its cause. Some of the nuclear DNA content variation has been explained by the presence of B chromosomes or knobs. However, variation in these two structures does not account for all of the observed variation. In order to identify other fluctuating DNA sequences, a rapid and reliable method of estimating relative abundance of DNA sequences needed to be developed. The potential of flow cytometry in conjunction with spot hybridization for determining relative abundance of specific DNA sequences in maize was studied. Different numbers of G1 phase nuclei were sorted on nitrocellulose filters and non-radioactive hybridization and signal detection performed. Results from these experiments revealed a significant, positive linear correlation between the amount of target sequence and signal density using both knob (R</it> = 0.98) and ribosomal spacer (R</it> =0.99) DNA sequences. In addition, G1 phase nuclei of eight inbred lines differing in the amount of knob heterochromatin, were sorted on to filters and the non-radioactive hybridization and signal detection performed. A significant, positive linear correlation between C-band number and signal density (R</it> =0.83; P</it> = 0.0051) as well as between per cent heterochromatin and signal density (R</it>=0.96;P</it> = 0.0002) was observed. These results indicate the usefulness of flow cytometry for spot hybridization in determining the relative abundance of DNA sequences in the maize genome.