Unique mtDNA restriction fragment in an S cytoplasm fertile revertant line retaining the S1 and S2 plasmids

Presence of S1 and S2 plasmids is characteristic of mitochondrial DNA of male sterile S cytoplasm maize. Male fertile revertants of S cytoplasm in most nuclear backgrounds typically have lost the S1 and S2 molecules; however, fertile revertants in the inbred Wf9 nuclear background still retain the S1 and S2 plasmids (L.J. Escote et al., MNL 59:100, 1985; MNL 60:127-128, 1986; T Ishige and B. Gengenbach, MNL 59:98-99, 1985; T. Ishige et al., MNL 60:126, 1986). In addition to the G', J' and R' fertile revertants of Wf9 we previously reported for S group materials from Japan, we also have obtained a similar S' fertile revertant line we observed as a spontaneously fertile plant among standard Wf9S plants grown at St. Paul in 1984. We report here our initial observations of mtDNA from this S' mutant.

S' mtDNA contains the S1 and S2 plasmids apparently unaltered (data not shown). Restriction digests of S' mtDNA with EcoRI revealed the presence of a ca. 6.5Kb fragment not visible in digests of the progenitor Wf9Srf3rf3 line or in several other sources of S cytoplasm materials either with or without the Rf3 allele. This 6.5Kb fragment was cloned into pUC119 and initially used as a hybridization probe to Southern blots of mtDNA (Fig. 1). Hybridization patterns for the intact 6.5Kb probe (unmarked left lane) were complex, but apparently identical, for 7 sterile S cytoplasm sources including Wf9Srf3rf3 from Minnesota (lane I), from Illinois (lane L), or from Japan (lane M); Wf 9SRf3Rf3 (lane J); A188Srf3rf3 (lane K); and putative progenitors (lanes D and F, respectively) for the R' and J' fertile mutants. The hybridization pattern for the S' mutant (lane H) was simpler with homology indicated to 2 mutant-specific fragments of ca. 6.5Kb and 4.4Kb. The R' (lane C), J' (lane E) and G' (lane G) mutants had differential hybridization patterns with strong homology either to 1 (R' and J') or 2 (G') mutant-specific fragments. N cytoplasm lines with W182BN (lane A) and Wf9 (lane B) nuclear genotypes exhibited hybridization patterns different from each other and from the S cytoplasm fertile mutant and sterile lines.

The cloned 6.5Kb fragment was hybridized to blots of clones containing the specific mitochondrial genes (atp-alpha, atp6, atp9, cob, coxI, coxII, coxIII, large rRNA, small rRNA) or other sequences (5Kb repeat of N cytoplasm, 1.94Kb plasmid, S1 and S2 subclones). Only the clone for atp9 showed significant homology to the 6.5 EcoRI cloned fragment from S'. The atp9 clone (a 2.2Kb XbaI fragment obtained from C.S. Levings) has a predicted protein coding sequence of 222bp, but the primary transcript in N cytoplasm is ca. 1.95Kb (R.E. Dewey et al., Proc. Natl. Acad. Sci. 82:1015, 1985). Figure 2 shows a restriction map of the 6.5Kb EcoRI fragment. A 1.2Kb XhoI-XbaI fragment from the 2.2Kb a1p9 clone when used as a probe showed the strongest hybridization to a ca. 600bp SmaI-XbaI fragment from the cloned 6.5Kb EcoRI fragment. The 2.2Kb XbaI clone of atp9 contains a BamHI site ca. 120bp from the terminal XbaI site which may also be present within the 600bp SmaI-XbaI fragment. Additional subcloning and transcriptional analyses are in progress to determine whether there are transcriptional differences related to atp9 in the fertile mutants and S sterile cytoplasm lines or whether the structural differences have no effect on gene expression.

Figure 1. Hybridization of the 6.5Kb EcoRI fragment to mtDNA from: A. W182BN (N); B. Wf9 (N); C. Wf9 (R'); D. Putative progenitor of Wf9 (R'); E. Wf9 (J'); F. Putative progenitor of Wf9 (J'); G. Wf9 (G'); H. Wf9 (S'); I. Wf9rf3rf3 (S) progenitor of Wf9 (S'); J. Wf9Rf3Rf3 (S); K. A188rf3rf3 (S); L. Wf9rf3rf3 (S) from Illinois (S. Gabay-Laughnan); M. Wf9rf3rf3 (S) from Japan (T. Ishige). Cloned DNA used as a probe is shown in the unmarked left lane. MtDNAs were digested with EcoRI and electrophoresed on an 0.7% agarose gel.

Figure 2. Restriction site map of the 6.5Kb fragment obtained from the S' fertile revertant. Homology to the atp9 gene was determined by probing with a 1.2Kb XhoI-XbaI fragment that contains the coding region of the gene.

Yunxia Wang and Burle Gengenbach

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