Polymorphism and replication of heterochromatic repeats in the DNA of Arabidopsis
The composition of the individual eukaryote's genome and its variation within a species remain poorly defined. Even for a sequenced genome such as that of the model plant Arabidopsis thaliana accession Col-0, the large arrays of heterochromatic repeats are incompletely sequenced. We defined by flow cytometry a set of A. thaliana accessions that differ in measured nuclear genome size. Using these geographically separate populations, we assayed variation in the heterochromatic repeat arrays using two independent methods and identified substantial polymorphism among them, with variation by as much as a factor of two in the centromeric 180 bp repeat, in the 45S rDNA arrays and in the Athila retroelements. In the accession with highest measured genome size, Loh-0, we measured more than a two-fold increase in 5S RNA gene copies relative to Col-0; results from fluorescence in situ hybridization with 5S probes were consistent with the existence of size polymorphism between Loh-0 and Col-0 at the 5S loci. Comparative genomic hybridization results of Loh-0 and Col-0 did not support contiguous variation in copy number of protein-coding genes on the scale needed to explain their observed genome size difference. We developed a computational data model to test whether the variation we measured in the repeat fractions could account for the different genome sizes determined with flow cytometry, and found that this proposed relationship could account for about 50% of the variance in genome size among the accessions. Our finding of a negative relationship between measured genome size and the copy number of centromeric repeats was unexpected, as centromeres may make up the single largest repeat class in the genome. Heterochromatic repeats have been demonstrated to be much less than fully replicated in the endoreplicated cells of some taxa. We investigated whether under-replication of repeated heterochromatic sequences in the higher ploidy nuclei of Arabidopsis distorts our genome size measurements or our assessment of the amounts of heterochromatin in the accessions. We found the under-replication amounts we observed with flow cytometry to be insufficient by a factor of five to account for the negative relationship between measured genome size and centromeric repeat amount.
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