Sequencing of mtDNA related to a recombination event involved with male fertility and/or toxin resistance in T cytoplasm

Restriction enzyme analysis of mitochondrial DNA (mtDNA) from normal (N), cytoplasmic male sterile (cms-T) and fertile revertants (regenerated from callus from immature embryos of cms-T) has identified a 6.6 Kb XhoI fragment that is unique to cms-T mitochondrial genome with only one exception (Brettell et al., Theor. Appl. Genet. 58:55, 1980; Gengenbach et al., Theor. Appl. Genet. 59:161, 1981; Umbeck and Gengenbach, Crop Sci. 23:584, 1983).

Comparative studies of the 6.6 Kb XhoI fragment from the cms-T mtDNA and the regions homologous to this fragment in the N genome and a fertile revertant, V3 (WF9T/W22 x A188Nrf x W22rf), provided us a clue to the molecular basis of male sterility and/or T-toxin sensitivity in T cytoplasm.

Southern analysis of cosmid clones containing the 6.6 Kb XhoI fragment from cms-T mtDNA and homologous regions from N and the fertile revertant V3 mtDNA show that a number of events are necessary to explain the formation of the 6.6 Kb XhoI fragment in cms-T and its subsequent loss in the fertile revertant. This includes the formation and the loss of a 4 Kb repeat and at least two independent recombinational events (Fauron et al., Curr. Genet., in press). The region of interest could be narrowed down to a 1.5 Kb AvaI fragment internal to the 6.6 Kb AvaI fragment. The 1.5 Kb AvaI fragment contains part of two reading frames, ORF13 and ORF25, identified by Dewey et al. (Cell 44:439, 1986). This 1.5 Kb AvaI fragment hybridizes to a 2.1 Kb AvaI fragment in N and V3 (Abbott and Fauron, Curr. Genet. 10:777, 1986). Fine mapping of the 2.1 Kb AvaI fragments of N and V3 shows that the two are almost identical (Fauron et al., in press). The data suggest that the two parts of a DNA sequence in N, that have been split and dispersed in T mtDNA, are brought back together through a recombination event in the V3 mtDNA, the fertile revertant.

Sequence analysis of those two 2.1 Kb AvaI fragments and the cms-T specific 1.5 Kb AvaI fragment has enabled us to localize the recombination site. The break point is located 6 bp past the ORF13 stop codon (Figure 1). Upstream from this point, the T sequence represents the 3' end of the ORF13 unique to cms-T. It is interesting to see that the recombinational mechanism that gave rise to the 2.1 Kb AvaI fragment in V3 has reconstructed an exact N-like sequence at the break point. Between the break point and the beginning of ORF25, the region seems very unstable as seen by the many changes between the 3 genomes.

Another fertile plant, V32, regenerated from a different embryo [WF9T/W22 x A188Nrf x A188Nrf], was sensitive to T toxin and produced four seeds [A,B,C,D] of which A gave a partly sterile and toxin sensitive plant and D a fertile toxin resistant plant. Interestingly V32A, V32B mtDNA contains the 1.5 Kb AvaI fragment while the V32C, V32D mtDNA contains the 2.1 Kb AvaI fragment.

Figure 1.

C. Fauron, J. Qin, A. Abbott and R. Brettell

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