Many studies have now established that there is a great deal of mitochondrial DNA variability detectable by restriction enzyme analysis in a wide range of organisms. In maize, examples of restriction endonuclease fragment differences have been found among the male sterile cytoplasms (Pring and Levings, Genetics 89:121; Borck and Walbot, Genetics 102:109), Mexican races of maize (Kemble et al., TAG 65:120), North American inbred lines (Levings and Pring, J. Hered. 68:350; McNay et al., PMB 2:177), and relatives of maize (Sederoff et al., PNAS 78:5953; Timothy et al., Maydica 28:139). Much of the observed variation is attributable to complex rearrangements rather than to simple base changes leading to loss of particular restriction sites (McNay et al.; Sederoff et al.). Considering the existence of this widespread variability, we wished to examine the relative stability of mitochondrial DNA in individuals from a single inbred line over short time spans: Is the mitochondrial DNA in a particular inbred line homogeneous? Lonsdale et al. (in: Plant Molecular Biology, R. Goldberg, ed., p. 445) have proposed that mitochondrial genomes may exist in multiple forms, which would arise by intramolecular recombination. By self-pollinating maize plants to allow them to sort out any pre-existing variability or to differentially amplify and/or segregate multiple molecular forms, do we begin to see changes within the mitochondrial DNA restriction enzyme fragment patterns? Can we detect rapid changes over short periods of time within a single inbred line? While many generations might be required to sort out pure clones, differences in the stoichiometries of restriction enzyme fragments would be expected to be detectable as differentially staining DNA bands.
The starting material used in our studies was bulk sib seed of the inbred B37N line, kindly provided by Pioneer. In 1981, plants grown from several kernels (probably derived from different ears) were self-pollinated and kernels from 6 of these ears, chosen at random, were grown in 1982 and again selfed. In 1983, mitochondrial DNA was isolated from 7 individual third generation plants and 3 sets of pooled third generation plants (see diagram), as well as from a mixture of the bulk B37N plants. These samples were subjected to restriction endonuclease digests using 5 enzymes: BamHI (5'GGATCC), XhoI (5'CTCGAG), PstI (5'CTGCAG), HindIII (5'AAGCTT, an AT-rich cutter) and SmaI (5'CCCGGG, a GC-rich cutter). After electrophoresis on 0.7% agarose gels, the DNA bands were stained with ethidium bromide and photographed. A total of 199 fragments were resolved from each individual; this represents a test of about 0.2% of the approximately 600 kb maize mitochondrial genome for the presence of a particular nucleotide sequence. Upon visual inspection, no mobility differences in restriction fragments could be detected either between individuals with the same maternal parent or among all of the individuals examined. The stained gels gave good resolution of the fragments between 26 and 1.5 kb. Based on standard curve measurements, a change of 75 base pairs would have been detectable in the 1.5 kb range, and changes of 500 bases would have been distinguishable in the 26 kb region. Therefore, if rearrangements, large deletions/insertions or recombinational changes were occurring, they would have been detected in this test. Yet no change was seen. In addition, no differences in band stoichiometries were seen. The mitochondrial genome of the inbred B37N line appears to be very stable.
Thus, despite the variability observed in mtDNA between lines with normal cytoplasms, we have detected no variability between individuals of a single inbred line after three generations of separating cytoplasmic lineages. If there were any mtDNA polymorphisms, three generations may not have allowed sufficient time to sort them out. However, in developing stable inbred lines, breeders probably selected for stability of mitochondrial genomes and also for nuclear/cytoplasmic compatibility. Our results do suggest that, if mtDNA assumes multiple molecular forms, mechanisms must exist to ensure similar distributions during cell divisions. We suggest that mtDNA changes in maize, such as those seen between inbred lines (which appear to result from genomic rearrangements), are rare and are possibly mediated by nuclear genes.
Tony Oro and Kathleen Newton
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