In contrast with animal mitochondrial DNA (mtDNA), which is typically 10 megadaltons (Md) per mitochondrial genome, plant mtDNA is far more complex (Stadler Symp. 10:77-94). The maize mitochondrial genome has been estimated to be 320 Md (B. L. Ward, R. S. Anderson and A. J. Bendich, personal communication). In addition to larger size, plant mtDNAs are characterized by molecular heterogeneity (in Extrachromosomal DNA: ICN-UCLA Symposium on Molecular and Cellular Biology 15:63-73, Academic Press), observed as classes of circular chromosomes which vary in size, relative abundance and sequence (Devel. Genetics 1:363-378). In order to learn about the molecular events which have occurred in the evolution of these unusual DNAs, we have directed our attention to the mtDNA of maize and its closest relatives, the teosintes.

Three races of teosinte have been chosen for this analysis, based on systematic relationships and diversity of the restriction patterns of the mtDNA (PNAS 76: 4220-4224). Races Central Plateau (CP), Guatemala (GU) and perennial teosinte (ZP) were selected. CP is closely related to maize and was selected as a representative of annual teosintes. GU and ZP were selected because their mtDNAs are distinct from each other and each differs greatly from maize and the other teosintes.

Restriction enzyme analysis has been combined with DNA transfer techniques and molecular hybridization to follow molecular events which have occurred in the evolution of mtDNA. We have used cloned DNA fragments from normal (N) maize mtDNA as labeled DNA probes to detect the presence and size of specific sequences in restriction digests. Following electrophoresis and DNA transfer (using either nitrocellulose or DBM paper) labeled DNA from cloned probes was hybridized to the restriction digests. The number and size of the fragments with sequence homology were detected by autoradiography.

When single cloned fragments from N mtDNA are labeled by nick translation and hybridized to teosinte mtDNA, a complex variety of patterns appears. The results indicate a far more complex mode of evolution in mtDNA of maize and its relatives than has been observed in mtDNA of animals.

Twenty-three cloned fragments of N mtDNA were selected for this study. Each clone contains a single fragment of N mtDNA. All clones showed homology with specific fragments in all three races of teosinte, indicating general conservation of homologous sequences. Eight clones hybridized identically to N mtDNA and all three races of teosinte. However, most clones hybridized to bands of teosinte mtDNA which varied in position, number or intensity.

Only one cloned fragment demonstrated a change in teosinte which indicated a change in a single restriction site. In this case, clone 542, two bands appeared in GU and ZP which sum to the molecular weight of the fragment in N and CP. This type of change is typical of those observed in animal mtDNA (PNAS 77:3605-3609) but is relatively rare in the species examined in this study. Most patterns observed were more consistent with rearrangements rather than with site mutations.

The simplest kinds of rearrangement seen are inversion-type changes and insertion-deletion type changes. Other patterns observed are so complex that it would be hard to retain sequence homology if base substitution mutation were sufficiently extensive to generate such changes. DNA methylation could potentially result in patterns which could mimic both site changes and rearrangements, but extensive studies have shown that methylation does not occur to such an extent in mtDNA (B. L. Ward, R. S. Anderson and A. J. Bendich, personal communication; PNAS 77:6415-6519).

An interesting class of variation observed is that of intensity variants. In these cases, a given band is present in both species, but is greatly reduced in intensity. We propose that the restriction fragment has changed its relative abundance in the mtDNA because different classes of circular DNAs are known to vary in relative abundance (in Extrachromosomal DNA, Academic Press). Recombination or rearrangements involving different circle classes could generate a change in the abundance of a specific restriction fragment.

Our results indicate that the models of mtDNA evolution used for animal systems (PNAS 76:5269-5273) do not apply to plant mtDNA since rearrangements feature prominently in the evolution of sequence organization.

R. R. Sederoff, C. S. Levings, III, D. H. Timothy and W. W. L. Hu

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