Quantitative analyses of D. mauritiana mitochondrial genome based on restriction cleavage

For lines heteroplasmic for D. mauritiana wild-type mtDNA and D. melanogaster wild-type mtDNA, the sequence difference between the two genomes did not allow performance of qPCR. Thus, we quantified the relative amount of genomes distinguished by restrictions enzyme cleavage site by comparing the ratios of diagnostic restriction fragments. Total DNA was collected from 40 adult flies of various heteroplasmic lines from generation 1 to generation 4 (for generation 0, total DNA was collected from the mother after it was mated with males at 25 °C for two days to lay eggs). A mtDNA region (mt11517 – 12529) was amplified by PCR (30 cycles of 95 °C 30 s, 50 °C 30 s and 60 °C 60 s). The PCR products were then digested completely with XhoI under the conditions recommended by the supplier (NEB). The digested DNA was separated by gel electrophoresis, and the ratio of cut and uncut DNA was estimated by measuring the intensity of bands using ImageJ.