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Mitochondrial DNA in Molecular Systematics Mitochondria - organelle found in eukaryotic cells - cellular respiration – ATP production Mitochondria Evolution • Endosymbiotic Theory – Ivan Wallin (1920s) and Lynn Margulis (1981). • Proto-Eukaryotic cell incorporated a protobacterial cell and formed a symbiotic relationship (a billion years ago). cyanobacteria Primordial eukaryotic cell Eukaryotic cell Perform Symbiotic Relationship mtDNA • mtDNA is maternally inherited in animals and plants. • More than 1300 complete mitochondrial sequences have been generated. • Most of the mitochondrial complete sequences are belonged to animals and algae. • Only a few plant species have their mitochondrial genome sequenced: Nicotiana tabacum, Oryza sativa, Triticum aestivum, Zea mays, Sorghum bicolor, Marchantia polymorpha. • Plant mtDNA is far more complex than animal mtDNA – larger size (300-600kb) and variable in size (up to 2000kb). Comparison of Plant mtDNA and cpDNA Plant species cpDNA mtDNA Nicotiana tabacum Oryza sativa Triticum aestivum Zea mays Sorghum bicolor Marchantia polymorpha 156kb 135kb 135kb 140kb 141kb 121kb 431kb 492kb 453kb 570-680kb 496kb 187kb Plant mtDNA • In addition to larger size, plant mtDNA are characterized by molecular heterogeneity. • Large duplications are readily created and lost. Plant mtDNAs contain at least one large (1-14kb) repeated sequence. There is no pattern to the sequences (including genes) that are duplicated in the mitochondrial genomes of different plants. Plant mtDNA • Recombination between repeats creates a complex, multipartite genome structure. All of the large repeats found in plant mtDNAs appear to be engaged in high-frequency inter- and intra-molecular recombination. Plant mtDNA Tricircular structure of the Brassica campesteris mitochondrial genome Plant mtDNA • mtDNA contains short dispersed (50-1000bp) repeats scattered throughout the genome. • mtDNA contains many foreign sequences. cpDNA sequences of all kinds (labelled as C111), some as large as 12kb in length, are found integrated in plant mtDNA. Plant mtDNA • Plant mtDNAs change very slowly in nucleotide sequences. Rates of nucleotide substitutions are 3-4 times lower in plant mtDNA than in cpDNA, 12 times lower than in plant nuclear DNA, and 40-100 times lower than in animal mtDNA. • Plant mtDNAs rearrange very rapidly. No two eximined species of flowering plants have the same gene order. Even closely related species differ by one or a few large inversions, whereas the genomes of more distantly related species are virtually randomized with respect to sequence arrangement. Animal mtDNA • Animal mtDNAs are relatively smaller than the plant mtDNAs. • The genome size is more conserved. Comparison Genome Size of Animal mtDNA Animal species mtDNA Aedes aegypti Alligator sinensis Apis mellifera Boa constictor Elephas maximus Gallus gallus Homo sapiens Octopus ocellatus Pongo pymaeus Rana nigromaculata 16.7kb 16.7kb 16.3kb 18.9kb 16.9kb 16.8kb 16.6kb 16kb 16.4kb 17.8kb Human Mitochondrial Genome Human mtDNA composes of a control region (CR), genes encoding 2 rDNAs (12S and 16S), 22 tRNAs (open circles), 13 polypeptides. Animal mtDNA • Non-recombination. • Contains less non-coding sequences. • Higher base substitution rate (even higher than the nuclear DNA regions). • Gene order/structure is more conserved (stable). mtDNA in Plant Systematics • Mitochondrial DNA regions commonly used for plant systematics: coxI, nad2, atpA, cob, coxIII, 18S, 26S etc. • The high rates of rearrangements and low rates of point mutations make mtDNA essentially worthless for the restriction site-based reconstructions of intrafamilial phylogeny for which cpDNA is so well suited. • The occasional losses of mitochondrial genes and introns may also serve as useful markers of phylogeny. • The low rate of mtDNA substitutions suggests that comparative sequencing efforts will be most rewarding at higher phylogenetic levels. mtDNA in Animal Systematics • RFLP on the whole mitochondrial genome is common in molecular systematics in animals. (involves the isolation of mtDNA from total DNA) • DNA sequencing is done on variable regions such as D-loop region (control region), cytochrome b, cytochrome oxidase I, cytochrome oxidase III, 16S, 12S etc.