A. Bashir et al., DETERMINING RELATIVE ABUNDANCE OF SPECIFIC DNA-SEQUENCES IN FLOW CYTOMETRICALLY SORTED MAIZE NUCLEI, Journal of Experimental Botany, 46(285), 1995, pp. 451-457
A wide breadth of DNA content variation has been reported among maize
lines. White the extent of this variation has been well documented, fe
w studies have focused on its cause. Some of the nuclear DNA content V
ariation has been explained by the presence of B chromosomes or knobs.
However, variation in these two structures does not account for all o
f the observed variation. In order to identify other fluctuating DNA s
equences, a rapid and reliable method of estimating relative abundance
of DNA sequences needed to be developed. The potential of flow cytome
try in conjunction with spot hybridization for determining relative ab
undance of specific DNA sequences in maize was studied. Different numb
ers of G1 phase nuclei were sorted on nitrocellulose filters and non-r
adioactive 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 kn
ob (R=0.98) and ribosomal spacer (R=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 hyb
ridization and signal detection performed. A significant, positive lin
ear correlation between C-band number and signal density (R=0.83; P=0.
0051) as well as between per cent heterochromatin and signal density (
R=0.96; P=0.0002) was observed. These results indicate the usefulness
of flow cytometry for spot hybridization in determining the relative a
bundance of DNA sequences in the maize genome.