ISOLATION AND CHARACTERIZATION OF X-CHROMOSOME-DERIVED DNA-SEQUENCES FROM A DIOECIOUS PLANT MELANDRIUM-ALBUM

A number of X chromosome DNA sequences have been isolated from a dioec ious plant, Melandrium album (syn. Silene latifolia), using chromosome microdissection followed by degenerate oligonucleotide-primed polymer ase chain reaction (DOP-PCR) amplification. Six DNA clones were select ed and further characterized by DNA/DNA hybridization techniques to ch eck their copy numbers, sex-specific methylation patterns, species spe cificity and positions on chromosomes. These clones were moderately to highly repetitive (approximately 10(3)-10(5) copies per haploid genom e) and none of them gave a positive signal on Northern blots. One of t he clones yielded a sex-specific methylation pattern: its abundant non -methylated CCGG island was found only in males. All the clones also h ybridized to two closely related dioecious Melandrium species (M. rubr um and M. dicline). Nucleotide sequences of two X-derived clones showe d a number of internal short direct repeats; one of them strikingly re sembled a plant conservative telomere sequence (TTTAGGG). None of the clones hybridized to the X chromosome only, but all were localized at the telomeric heterochromatic regions (DAPI C-bands) of both arms of a vast majority of M. album chromosomes using the fluorescence in situ hybridization (FISH) technique. However, the non-homologous arm of the Y chromosome (contrary to the arm homologous to the X chromosome, pos sessing the pseudoautosomal region) showed neither a DAPI C-banding-st ained heterochromatin nor a FISH signal with any of the DNA probes tes ted, thus indicating its evolutionary diversification.