In animals studied to date, the maternally inherited mitochondrial DNA (mtDNA) evolves about 5-10 times faster than the nuclear genome (Brown et al., PNAS 76:1967), based on detection of changes in restriction endonuclease patterns. These differences, indicative of base changes and deletions/insertions in mtDNA, can be readily used to distinguish species. In higher animals, the mtDNA contains a hypervariable region near the origin of replication; analysis of restriction fragment and sequence changes in this region can be used to distinguish individuals from each other. In one study of bovine mtDNA inheritance, a single Holstein had granddaughters differing at one restriction site (Hauswirth and Lapis, PNAS 79:4686).
In the preceding note Oro and Newton showed that no variations in restriction fragment mobility or band stoichiometries were detectable in individuals of a single inbred line after three generations of selfing. In evaluating these data, one problem is that the time-frame for the variation found in the maize mtDNA organization is unclear; that is, we do not know the time of divergence of N and the male sterile cytoplasms nor the age of existing lines of maize, so no calculation of the rate of change is possible.
However, these data can also be used to set an upper limit to the generation of new restriction site polymorphisms arising as the base changes in the sites examined in the B37N mtDNA, using the model of Nei and Li (PNAS 76:5269) for estimating DNA divergence in a population. This model assumes random base-pair substitutions and estimates the time-frame of variability given the number of fragment changes in a population over time. Although we saw no changes, for the sake of calculations we assume 1 fragment change out of the 199 fragments detected in 5 restriction digests of a particular individual over three generations. Using the formula (Nxy/No)2 = e-2lrt where Nxy = number of fragments in common at time t, No = number of fragments in common at time 0, 2lt = rate of nucleotide substitution per nucleotide per unit time, and r = number of bases recognized by the restriction endonucleases used, we calculate a rate (2lt) of 8.4 x 10-4 (variance 0.5 x 10-4) base changes per nucleotide in 3 generations. Assuming a 600 kb genome size for maize mtDNA, we calculate that this rate of substitution represents a change of less than 504 nucleotides (variance 30 bases) per generations. Because we have, in fact, detected no changes thus far, this rate of substitution is an upper limit to the variation in maize mtDNA over time. The upper limit of variation in maize mtDNA over several generations is low in comparison with the rate of actual base substitution in animal mtDNA. For example, in a survey of populations of higher primates the rate of nucleotide substitution in mtDNA was 3.2 x 10-2 per year (Brown et al., PNAS 76:1960), forty-fold higher than the upper limit in maize.
Rates of substitution per year in animals are calculated from surveys of existing differences within the whole species or among subpopulations, and from knowledge of the time-frame of speciation from the fossil record. This type of data cannot be obtained for maize because of the paucity of fossils by which to date the emergence of species in higher plants.
We speculate that the changes observed among N, T, C and S mtDNAs and among inbred lines are most likely due to rearrangements or insertion/deletion events rather than a rapid rate of nucleotide substitution in restriction sites.
Tony Oro and Virginia Walbot
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