|Riepsamen, Angelique -|
|Gibson, Tracey -|
|Rowe, Janet -|
|Subbotin, Sergei -|
|Dowton, Mark -|
Submitted to: Journal of Molecular Evolution
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: November 19, 2010
Publication Date: December 16, 2010
Repository URL: http://ddr.nal.usda.gov/dspace/handle/10113/49430
Citation: Riepsamen, A.H., Gibson, T., Rowe, J., Chitwood, D.J., Subbotin, S.A., Dowton, M. 2011. Poly(T) variation in heteroderid nematode mitochondrial genomes is predominantly an artifact of amplification. Journal of Molecular Evolution. 72(2):182-192. Interpretive Summary: Nematodes are microscopic worms that attack plant roots and annually cause ten billion dollars of crop losses in the United States. A major problem with reducing nematode-induced crop losses is that safe and effective control measures for nematodes are not available. One approach to developing new control methods is to disrupt unusual biochemical features of nematodes; the search for such features has previously revealed unusual aspects in the structure of a certain type of DNA called mitochondrial DNA. In this paper, an international team of scientists demonstrate that one such unusual aspect of mitochondrial DNA in several nematode species actually results from artifacts caused by analytical procedures and does not indicate a true biological phenomenon. The results are significant because they provide the first warning to scientists that nematode mitochondrial DNA analysis is prone to error and that diligent caution must be invoked when conducting experiments. Therefore, the results will be used by the large numbers of scientists investigating the biochemistry of nematode DNA.
Technical Abstract: We assessed the rate of in vitro polymerase errors at polythymidine tracts in the mitochondrial DNA (mtDNA) of a heteroderid nematode (Heterodera cajani). The mtDNA of these nematodes contains unusually high numbers of poly(T) tracts, and has previously been suggested to contain biological poly(T) length variation. However, using a cloned molecule, we observed that poly(T) variation was generated in vitro at tracts >6 Ts. This artifactual error rate was estimated to be 0.000073 indels/poly(T) tract >6 Ts/cycle. This rate was then compared to the rate of poly(T) variation detected after the amplification of a biological sample, in order to estimate the ‘biological + artifactual’ rate of poly(T) variation. There was no significant difference between the artifactual and the artifactual + biological rates, suggesting that the majority of poly(T) variation in the biological sample was artifactual. We then examined the generation of poly(T) variation in a range of templates with tracts up to 16 Ts long, utilizing a range of Heteroderidae species. We observed that T deletions occurred 5 times more frequently than insertions, and a trend towards increasing error rates with increasing poly(T) tract length. These findings have significant implications for studies involving genomes with many homopolymer tracts.