Evidence of chloroplast and mitochondrial DNA variation among male-sterile cytoplasms

Sources of cytoplasmic male sterility in maize have been classified into the T, C, and S groups, based on fertility restoration patterns in a variety of inbred lines. We previously (Science 193:158-160) reported that the T and N mitochondrial DNAs (mtDNAs) are distinct, using restriction endonuclease fragment analysis as an assay. These studies also showed that the mtDNA of the N recurrent parent is not inherited paternally. The present study was designed to examine the chloroplast and mitochondrial genomes of the three major sterility groups and of normal cytoplasms. MtDNAs from five lines in T cytoplasm, three lines in C cytoplasm, four lines in S cytoplasm, and seven lines in N cytoplasm were prepared. Cytoplasms EK, H, SD, and CA, members of the S group, were included. Chloroplast DNAs (ctDNAs) from most of the lines were also examined.

The DNAs were purified by cesium chloride-ethidium bromide preparative ultracentrifugation, and upper (linear and open circular DNA) and lower (supercoiled DNA) zones were collected. Routine analyses were conducted with the former since no apparent differences in fragment patterns were found between the two fractions. Restriction endonuclease Hind III and Bam I were used to digest the DNAs, and fragments were electrophoresed in 1.0% agarose gels in Tris-phosphate-Na2EDTA buffer. Restriction patterns of mtDNAs are quite complex, with 40-50 bands produced by each enzyme. A schematic diagram of the Hind III patterns is shown in Fig. 1. The fragment patterns clearly indicate that the mtDNAs of the N, T, C, and S cytoplasms are distinct. Bam I also clearly distinguished the four cytoplasms. Characteristic patterns of the T, C, and S cytoplasms were the same regardless of nuclear background. Cytoplasms H, EK, CA, and S gave Hind III patterns indistinguishable from each other and from S cytoplasm mtDNA, even though these cytoplasms have been reported to be different in terms of fertility restoration patterns. There was some variation among N cytoplasm mtDNAs, but the magnitude of their variation was much less than observed for the male-sterile cytoplasms.

The molecular weight of mtDNA was about 108, with some apparent variation among cytoplasms. This estimate is tentative in that the patterns are too complex with these enzymes for accurate molecular weight determinations. Interestingly, the C cytoplasm was more closely related to the N cytoplasm than were the S and T cytoplasms, using the molecular weights of common Hind III fragments as an index of relatedness. The T cytoplasm was most divergent of the three male-sterile cytoplasms, suggesting a possible basis for the susceptibility of T cytoplasm to B. maydis and P. maydis. Fragment patterns of mtDNA of all members of the S group are complicated by the presence of two unique circular DNAs (see Levings, et al., this issue). These DNAs were restricted by the endonuclease and contributed low molecular weight fragments to the S patterns.

Figure 1 (left) and Figure 2 (right).

In contrast to mtDNA, maize ctDNA restriction patterns with Hind III are quite simple (Fig. 2). Only 25 bands were observed among the N, T, C, and S cytoplasms, with several occurring in double frequency. No variation was found among the N, T, and C cytoplasms, while a slight shift of one band (3.6 - 3.8 cm in Fig. 2) was observed in the S cytoplasm. Cytoplasms H, EK, SD, and CA also were characterized by this single band shift. This slight displacement of one band in the S cytoplasm ctDNA suggests lack of paternal inheritance of the N ctDNA. The molecular weight of maize ctDNA was 7.5 - 8.0 x 107, which is similar to estimates made by other criteria.

These results imply that cytoplasmic genome variation, especially for mtDNA, can be demonstrated among male-sterile cytoplasms. The cytoplasmic genomes appear to be not inherited through the pollen, although our assays probably would not detect 5% contamination. Restriction patterns of mtDNAs also indicate that the genome is conserved among nuclear backgrounds from diverse geographical areas; T cytoplasm, for example, was examined in lines W64A, B37 x NC236, T204, NC7 x T204, and F44. Variation of mtDNA among the T, C, and S cytoplasms is compelling evidence of mitochondria as probable carriers of factors conditioning cytoplasmic male sterility, but the evidence should be interpreted as circumstantial. Failure to distinguish ctDNAs among the N, T, and C cytoplasms with one restriction endonuclease does not prove that the genomes are identical, nor unrelated to the sterility-susceptibility traits. Other kinds of evidence will be required to effectively establish the nature of the cytoplasmic agent(s) involved.

D. R. Pring* and C. S. Levings III

*ARS, USDA and Department of Plant Pathology, University of Florida


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