DNA restriction endonuclease cleavage and Southern hybridization analysis of maize ribosomal DNA were undertaken to determine if large scale restriction site polymorphism or repeat length heterogeneity can arise in regenerated plants due to the tissue culture cycle. Nuclear DNA from 25 regenerated plants and one seed-grown plant (control) of the maize inbred A188 were subjected to restriction analysis by cleavage with EcoR1, BamH1, and Sst1, and hybridized with a nick-translated probe of the maize 9kb ribosomal repeat. The plants used in this analysis were regenerated from a single tissue culture line produced over a period of 22 months. In addition, the cell culture pedigree, that is the cell lineage relationship of each regenerated plant to each other plant, was maintained.
The maize inbred A188 contains approximately 7,000 copies of the ribosomal repeat per 2C nucleus. Based on a series of reconstruction experiments, a large scale change such as variation in the length of the external spacer could be detected if approximately 100 copies (1.43% of the total number of repeats) had the occurrence of a common change. A new site dividing the 9kb fragment into two 4.5kb fragments would be detected if 200 copies of the 4.5kb ribosomal fragment were present in the new band. This would stoichiometrically correspond to a total of 100 copies of the rDNA repeat. The limits of other detectable variation would be dependent on the number of site changes that occurred in common and the DNA fragment size variation that those changes produced. Overall, no large scale variation in the rDNA genes isolated from the 25 regenerated plants was detected by this analysis. This implies that these genes appear to be stable in the regenerated plants over a 22 month period of growth in tissue culture.
Southern hybridization of the BamH1 digest, however, showed variation in the amount of DNA present in the three different restriction size fragments produced by this endonuclease. There are two BamH1 sites located in the maize ribosomal repeat. Cleavage of the ribosomal repeat at the restriction site present in the 18S gene results in a 9kb fragment. Site heterogeneity, believed due to methylation of the BamH1 site located in the 26S rDNA gene, is responsible for the presence of the 5 and 4kb bands. Approximately 50% of the 26S BamH1 sites are believed to be methylated, resulting in an even distribution of the ribosomal DNA between the 9kb and the combined 5 and 4kb fragments. A decrease in methylation would be detected by a decrease in intensity of the 9kb band and a corresponding increase in the intensity of the 5 and 4kb bands in each sample. Such variation was detected in densitometer scans of the BamH1 digest. The extent of this variation observed in the 26 samples showed a distribution around a 9kb fragment mean of 55% with a standard deviation of 8%. The range about the mean is from 41 to 68% for the 9kb fragment. The level of variation observed in this analysis does not show a correlation of culture age to restriction site heterogeneity. In addition, there is no correlation of tissue culture pedigree to DNA fragment distribution. Further, the variation fits a normal distribution about the mean (based on a Wilk-Shapiro W statistic) and therefore appears to be random. If the variation observed was due to a directed genetic change, such as a reduction in site-specific methylation, the data would not be expected to be normally distributed about the mean. Because these measurements do not show discrete differences in the density of the different DNA fragment size classes, and the data approximate a normal distribution, the observed differences may be due to physiological variation or experimental error and not genetic variation. These data do, however, indicate the possibility of random changes in the cleavage heterogeneity of this BamH1 site due to the tissue culture cycle.
Gary Benzion*, Brenda G. Hunter, Irwin Rubenstein and Ronald L. Phillips
*Department of Agronomy, University of Kentucky, Lexington, KY
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