Mitochondrial DNA (mtDNA) is unusual in its rapid rate of evolution and high level of intraspecies sequence variation. The latter is thought to be related to the strict maternal inheritance of mtDNA, which effectively isolates within a species mitochondrial gene pools that accumulate mutations and vary independently. A fundamental and as yet unexplained aspect of this process is how, in the face of somatic and germ-line mtDNA ploidy of 10(3) to 10(5) (refs 4, 5), individual variant mtDNA molecules resulting from mutational events can come to dominate the large intracellular mtDNA population so rapidly. To help answer this question, we have determined here the nucleotide sequence of all or part of the D-loop region in 14 maternally related Holstein cows. Four different D-loop sequences can be distinguished in the mtDNA of these animals. One explanation is that multiple mitochondrial genotypes existed in the maternal germ line and that expansion or segregation of one of these genotypes during oogenesis or early development led to the rapid genotypic shifts observed.

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Two polymorphic mitochondrial DNA genomes, differing by a single Hpa II restriction site, are present at significantly different levels in tissue of three sibling dairy cows. The relative ratio of the two heteroplasmic molecules varies 3-fold among these three animals and documents a rapid segregation of mitochondrial genotypes in mammals. DNA sequencing shows the difference is due to a single guanine at position 364 in bovine mitochondrial DNA. A model involving unequal partitioning of the two ampliflied mitochondrial DNA species during the early cell divisions of the embryo can explain the appearance of such variation in heteroplasmic sibling animals. The model provides a basis for understanding the rapid DNA sequence variation observed in vertebrate mitochondrial DNA despite its high copy number and strict maternal inheritance.The mechanism of inheritance of mitochondrial DNA (mtDNA) in vertebrates is poorly understood. It appears to be maternally inherited in all higher eukaryotes (1-4), although the molecular basis ofthis restriction is unknown. The mechanism of inheritance must accommodate this transmission bias and should account for the numerous sequence differences present between the mtDNA of individuals within a species, as well as between the mtDNAs of closely related species (5)(6)(7)(8). The rate of sequence change has been estimated to be at least 10 times faster than the rates of nucleotide sequence change in mammalian nuclear genes (8); this rapid sequence divergence occurs in the context of a high ploidy of mtDNA in both somatic and germ-line cells (9-12). We previously reported (13-16) Hpa II site is therefore an unambiguous marker for following the segregation of heteroplasmic mitochondria. Two siblings in this Holstein lineage are shown to contain approximately equal proportions of these two mtDNA species, whereas a significant difference is found in the level of heteroplasmy in a third sibling. This suggests that polymorphic mtDNA may partition unequally during either female germ-line development or early embryogenesis to yield progeny with different levels of heteroplasmy. Such a process would rapidly lead to fixation and hence mitochondrial gene evolution. MATERIALS AND METHODSAll of the animals described were Holstein cows registered with the Holstein-Fresian Association of America and were born, bred, and maintained in the Dairy Research Unit herd of the University of Florida (Gainesville). Record keeping and factors affecting the H15 pedigree accuracy have been discussed (13-15). Due to herd management and the time over which this pedigree has developed, a number of animals had been culled and were no longer available for analysis when this study was begun, including H15 (pedigree founder, purchased February 15, 1955, died June 6, 1966, and H308 (born October 25, 1969, died September 1, 1978. mtDNAs from brains or livers of culled animals were prepared from purified mitochondria (19). All sequence coordinates refer to the bovine mtDNA sequence of Anderson et al. (20); the mtD...