Molecular and chromosomal characterization of repeated and single-copy DNA sequences in the genome of Dasypyrum villosum

Abstract

Restriction fragment length polymorphism of ribosomal DNA repeated unit and single-copy DNA fragments and chromosomal distribution of a highly repeated sequence, have been studied to assess molecular markers and the extent of their heterogeneity in Dasypyrum villosum. Substantial variation has been found for the length of the intergenic spacer of ribosomal genes clustered in different alleles at Nor- VI locus of heterozygous individuals, but not within the cluster of rDNA of homozygous individuals. After Southern blots and hybridization to an intergenic spacer probe, each cluster of rDNA was detected as a single band with at least four variants differing for the number of 130 bp subrepeats in the intergenic spacer. One recombinant plasmid contained a 2270 bp DNA insert from the D. villosum genome that upon Sph I restriction endonuclease digestion was cleaved in three 380 bp repeat elements and one 1090 bp fragment. When Southern blots of Sph 1 digested D. villosum DNAs of different genotypes were hybridized to the 32P-labelled 380 bp repeat, a distinct ladder consisting of multiples of a basic repeat unit of about 380 bp in length was revealed on autoradiograms. The in situ hybridization of the 3H-labelled 380 bp repeat element showed that one chromosome pair (7V) was not labelled. In the other pairs, silver grains remained clustered at or near the telomeres. Dot-blot hybridization analysis of DNAs from a range of diploid, tetraploid, and hexaploid Triticeae species indicated that the 380 bp repeated element was a specific feature of the D. villosum genome. Other cloned DNA sequences of D. villosum showed a large restriction length polymorphism and one was located on V chromosomes.