DNA rearrangement associated with Mutator-homologous sequences in non-Mutator somaclones

We have been examining the fate of DNA sequences homologous to the transposon family Robertson's Mutator during tissue culture and regeneration of non-Mutator maize lines. The inbred line W182BN and eleven somaclonal variants derived from it were given to us by Elizabeth Earle. Nuclear DNA from the inbred and somaclones were compared by restriction digest, Southern blotting and hybridization with Mutator probes. W182BN was found to have three types of Mu-homologous sequences, as we have described previously (V. Chandler et al., Genetics 114:1007, 1986; C. Rivin et al., MNL 61:53, 1987): 1) an endogenous element structurally like Mu2, 2) Mu termini unassociated with internal sequences, 3) 2 copies of internal sequences without associated termini. An example of the latter type of sequence, designated MRS-A for Mu Related Sequence, has recently been cloned and sequenced (Talbert and Chandler, submitted).

The somaclonal variants showed no fragment length differences when restriction sites that lie within the Mu-homologous sequences were examined. However, when the DNA was cut with enzymes that recognize sites outside the Mutator sequences, two types of differences were observed: In several somaclones there appear to be new termini-homologous bands, and in one of the somaclones (designated 2049) there is a fragment length polymorphism for one of the internal sequences. Segregation analysis of F2s from the 2049 x W182BN cross shows that this second type of change is not a transposition. We do not yet know about the terminal RFLPs, though it seems possible that these might act as independent transposons. The rearrangement occurring in the internal sequence of somaclone 2049 was mapped using probes from MRS-A and its flanking sequences (Fig. 1). The genomic restriction maps (Fig. 2) show that the Mu related sequence and sequences flanking it on one side are unchanged in the somaclone. However, a major rearrangement has occurred in the DNA adjacent to the other side, as the inbred and somaclone restriction maps are different for at least 10Kb.

Figure 1. Mutator-related sequence and flanking DNA probes. A clone of MRS-A and flanking sequences (pINT) derived from W23 was given to us by Luther Talbert and Vicki Chandler. Subclones used for genomic mapping are shown above the map of pINT.

Figure 2. Genomic restriction map of a Mutator-related sequence and flanking DNA from the inbred line W182BN and its somaclonal variant 2049. R: EcoRI, B: BglII, H: HindIII, T: Tth111, N: Ncol, E: BstEII, F: Hinfl, S: SstI.

Chee Harn and Carol Rivin

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