Cytoplasmic male sterile lines of maize are classified into three groups, T, C and S, based upon which nuclear genes restore fertility (Beckett, Crop Sci. 11: 724; Gracen and Grogan, Agron. J. 65:654). While field tests lead to conclusive classifications, they are time-consuming; therefore, efforts have been directed toward finding reliable molecular markers of cytoplasmic types. Recently a rapid classification method based upon the occurrence of low molecular weight mitochondrial DNA plasmids was proposed by Kemble and coworkers (Nature 284:565; Genetics 95:451). These plasmids may be seen after mitochondrial DNA isolated from a few seedling shoots is subjected to agarose gel electrophoresis without restriction endonuclease treatment. Pring et al. (PNAS 74:2904) had previously shown that mitochondrial DNA from S cytoplasms is readily distinguished by the prominent 6.2 and 5.2 kb linear S plasmids. Kemble et al. found that the mitochondrial DNAs from T cytoplasms lack a 2.35 kb linear plasmid present in C, S and normal (N) cytoplasms, and that C is distinguished by additional small plasmid bands of approximately 1.57 and 1.42 kb.

In examining N, T, S and C cytoplasms in B37 (obtained from Pioneer), as well as apparently normal cytoplasms from other inbred lines, we have found that misclassifications may result if the small plasmids are used to determine cytoplasmic type. With B37, the 1.57 and 1.42 kb "C" plasmids were not observed; hence, uncut B37C and B37N mitochondrial DNAs were indistinguishable. Following restriction enzyme digestion, these DNAs were characteristically different. In addition, the male sterility phenotype of B37C was confirmed by genetic tests in the field. On the other hand, the 2.35 kb plasmid is clearly missing in B37T plants and appears to be replaced by a smaller plasmid (approximately 2.2 kb).

When mitochondrial DNA from the fertile inbred Ky21 line was analyzed, it was also found to lack the 2.35 kb plasmid and to have the smaller plasmid reported to be characteristic of T mitochondria. Since Ky21 carries the nuclear restoration genes for all tested T, C, and S male steriles, the possibility was raised that Ky21 has T cytoplasm but is not sterile due to the presence of the dominant Rf1 and Rf2 fertility restoring genes. Genetic analyses will test this proposition: i.e., if Ky21 is cms-T and it is crossed by lines not carrying the nuclear restorers, male sterility should be observed as Rf1 and Rf2 segregate out. We are currently testing F2 and backcross progeny from crosses between Ky21 and B37N for sterile segregants. Other cooperators have not seen male steriles in F2 progeny of Ky21 crosses with maintainer lines (E. Coe, pers. comm.).

We have tested Ky21 for two other molecular correlations with cytoplasmic type: mitochondrial DNA restriction endonuclease digestion patterns (Pring and Levings, Genetics 89:121) and mitochondrially synthesized protein profiles (Forde et al., Genetics 95:443). The Ky21 mitochondrial DNA fragments resulting from digestion with BamHI and XhoI are much more similar to those of B37N than to B37T (or C or S). While several differences between B37N and Ky21 patterns exist, Ky21 lacks all the B37 T-specific bands, including the 6.6 kb XhoI band which has been strongly correlated with the cms-T trait (Gengenbach et al., TAG 59:161; MGCNL 56:140). Mitochondria isolated from cms-T plants synthesize large amounts of a 13,000 MW polypeptide, which is made only at very low levels, if at all, by mitochondria isolated from other cytoplasmic types (Forde and Leaver, PNAS 77:418). Tissue culture-induced fertiles originating from cms-T also synthesize very reduced levels of this 13 kD polypeptide, which strengthens the connection between its synthesis and the expression of the male sterility phenotype (Dixon et al. TAG 63:75). A preliminary study of protein synthesis in isolated Ky21 mitochondria indicates that they do not synthesize elevated levels of the T-associated polypeptide.

Thus, the cytoplasm associated with the Ky21 line would be classified as T on the basis of the rapid test of undigested mitochondrial DNA, due to the absence of a 2.35 plasmid. However, using restriction enzyme fragment and protein synthesis markers, Ky21 would not be considered cms-T. If the Ky21 cytoplasm is indeed normal, it suggests that, while the rapid test can give a useful preliminary diagnosis (especially for cms-S), certain mitochondrial DNA restriction enzyme fragments and mitochondrially synthesized polypeptides may be more reliable molecular tools with which to discriminate between N and cms-T.

Kathleen J. Newton

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