Variation among alfalfa somaclones in copy number of repeated DNA sequences

Repeated DNA sequences of alfalfa (Medicago sativa L.) somaclonal variants were analyzed to determine if changes in copy number had occurred during tissue culture. DNA clones containing highly repeated nuclear sequences from the diploid line HG2 (2x = 16) were slot blotted and probed with labeled DNAs from HG2 and several somaclones of HG2. Two DNA clones that differed visually in hybridization intensity among the plant DNAs and one clone that had constant hybridization intensity were selected and used as probes on Southern blots and slot blots containing equal quantities of DNAs from HG2 and 15 regenerants. Statistically significant differences were detected in the copy number of two anonymous DNA sequences initially selected as variable and in the copy number of sequences homologous to pea ribosomal DNA. Based on Southern blot analysis, these sequences appeared to be arranged as tandem repeats. The cloned sequence initially selected as stable did not vary significantly in copy number and it appeared to be arranged as a dispersed repeat. Both increases and decreases in copy number of repeated sequences were observed in plants from successive regeneration cycles. Results from this study indicate that specific repeated nuclear DNA sequences have changed copy number in plants regenerated from tissue culture.