5-Methylcytosine distribution and genome organization in Triticale before and after treatment with 5-azacytidine

Triticale (2n=6x=42) is a hybrid plant including rye (R) and wheat (A and B ) genomes, Using genomic in situ hybridization with rye DNA as a probe, we found the chromosomes of the R genome were not intermixed with the wheat ch romosomes in 85% of nuclei, After treatment of seedlings with low doses of the drug 5-azacytidine (5-AC), leading to hypomethylation of the DNA, the c hromosomes became intermixed in 60% of nuclei; the next generation showed i ntermediate organization. These results correlate with previous data showin g that expression of R-genome rRNA genes, normally suppressed, is activated by 5-AC treatment and remains partially activated in the next generation, The distribution of 5-methylcytosine (5-mC) was studied using an antibody t o 5-mC, Methylation was detected along the lengths of all chromosomes; ther e were some chromosome regions with enhanced and reduced methylation, but t hese were not located at consistent positions, nor were there differences b etween R and wheat genome chromosomes. After 5-AC treatment, lower levels o f methylation were detected, After 5-AC treatment, in situ hybridization wi th rye genomic DNA sometimes showed micronuclei of rye origin and multiple translocations between wheat and rye chromosomes. Genomic DNA was analysed using methylation-sensitive restriction enzymes and, as probes, two rDNA se quences, two tandemly organised DNA sequences from rye (pSc200 and pSc250), and copia and the gypsy group retrotransposon fragments from rye and wheat . DNA extracted immediately after 5-AC treatment was cut more by methylatio n-sensitive restriction enzymes than DNA from untreated seedlings. Each pro be gave a characteristic restriction fragment pattern, but rye- and wheat-o rigin probes behaved similarly, indicating that hypomethylation was induced in both genomes, In DNA samples from leaves taken 13-41 days after treatme nt, RFLP (Restriction Fragment Length Polymorphism) patterns were indisting uishable from controls and 5-AC treatments with all probes. Surprising diff erences in hybridization patterns were seen between DNA from root tips and leaves with the copia-fragment probes.