Mitochondrial DNA rearrangements associated with reversion of T cytoplasm to male fertility and disease resistance

Male-fertile plants resistant to Helminthosporium maydis race T have been regenerated from tissue culture of susceptible, male-sterile T cytoplasm maize (A188). Most lines derived from these plants carry altered mitochondrial DNA, as evidenced by restriction patterns. Common among these alterations is the rearrangement of a 6.6 kb XhoI fragment (Gengenbach, et al., Theor. Appl. Genet 59:161, 1981; Brettell, et al., MNL 56:13, 1982; Gengenbach and Umbeck, MNL 56:140, 1982). Eighteen of 19 lines examined to date carry the rearranged fragment. One interesting mutant, T-4 (Gengenbach and Umbeck, MNL 56:140, 1982), retains the intact 6.6 kb XhoI fragment and is not detectably different from parental T cytoplasm mtDNA, at least as detected by visual examination after restriction endonuclease digestion with several enzymes. This mutant was used for more extensive study. 1900-colony cosmid libraries of parental T and T-4 mutant mtDNAs were constructed in pHC79 and Grunstein-Hogness hybridizations were performed with a 6.5 kb BamH1 fragment of Wf9(N) mtDNA, which shares homology with the 6.6 kb XhoI fragment of T mtDNA (D. M. Lonsdale, personal communication).

Selected cosmids were isolated, hybridized, and mapped. Sequences of the 6.5 kb BamH1 fragment from N cytoplasm hybridized to the intact 6.5 kb fragment and to a BamH1 fragment of 9.0 kb in T cytoplasm. An XhoI fragment of 4.5 kb is internal to the 6.5 kb BamH1 fragment, while the 6.6 kb XhoI fragment is internal to the 9.0 BamH1 fragment. The 9.0 kb BamH1 and 6.6 kb XhoI fragment sequences share homology with the 6.5 kb BamH1 and 4.5 kb XhoI fragments. Mapping of these regions indicates that these fragments carry a recombination site; the two regions are co-linear to the recombination site, and diverge within the 6.5 and 9.0 kb BamH1 fragments. No apparent differences were observed between T and T-4 with BamH1 and XhoI. However, when a series of tetradeoxynucleotide-requiring restriction endonucleases were used on the isolated fragments, cosmids, or mtDNAs, Alul identified a rearrangement which distinguishes T-4 from T. The 6.6 kb XhoI fragment from T cytoplasm mtDNA hybridized to an AluI fragment of ca. 180 bp in T but not in T-4. Three A10 fragments of ca. 200, 650, and 1000 bp are shared by the 4.5, 6.5, 6.6,,and 9.0 kb fragments.

Lack of visual differences between restriction patterns of parental and mutant DNAs does not indicate that the DNAs are identical, as evidenced by the observations with AluI. We do not know if other regions of the genomes are rearranged, or if this alteration is causal to the male-fertile or disease-resistant phenotypes. We have probed mitochondrial DNA from T and T-4 with a series of cosmids from N and S mtDNA, and detect no other differences with BamH1 in about 250 kb of probed sequences. Based on these observations, therefore, we can only suggest that we have identified a small deviation which characterizes the T-4 mutant, which may or may not be associated with the phenotypes.

R. P. Wise, D. R. Pring, and B. G. Gengenbach

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