Year

2011

Abstract

Understanding the mechanisms by which mitochondrial genomes (mtDNA) evolve is essential to phylogenetics and population genetics. Animal mtDNA is typically assumed to be maternally inherited and absent of recombination events. If these assumptions are incorrect, i.e. if paternal mtDNA is also being inherited (termed paternal leakage) and/or recombination is occurring, the conclusions drawn from animal mtDNA analyses may be inaccurate. Recombination is typically detected by its products; recombinant mosaics of two differing „progenitor‟ molecules. However, maternal inheritance means recombinant products are indistinguishable from progenitor molecules, especially if the mtDNA population is homoplasmic. The majority of studies describe evidence of past recombination, where patterns of recombination are inferred by comparing the mtDNA of different individuals. Few studies assess contemporary mtDNA recombination, where recombinant molecules are observed as direct mosaics of known progenitor molecules. It has been suggested that the contact zones of hybridising species may be associated with paternal leakage, creating a heteroplasmic environment that facilitates recombination. Here we used populations of the potato cyst nematode, Globodera pallida, as a model organism to investigate past and contemporary mtDNA recombination, and paternal mtDNA leakage. G. pallida has demonstrated evidence of mtDNA recombination. The mtDNA is multipartite, comprising several unique small circular mtDNA (scmtDNA) molecules with an overlapping genetic organisation – indicative of recombination. Further, a ~3.4 kb non-coding scmtDNA region of G. pallida demonstrated significant genetic variation between populations, permitting analysis of recombination and paternal leakage between these populations. To assess past mtDNA recombination, this ~3.4 kb mtDNA region was analysed in 5 divergent G. pallida populations using recombination detection software. Evidence of past recombination was detected between a South American population and several European populations of G. pallida, as well as between two South American populations. This suggests that these populations may have interbred, paternal leakage occurred, and the mtDNA of these populations subsequently recombined. To assess the potential for this to occur, paternal leakage and contemporary recombination were assessed in the ~3.4 kb mtDNA region in the progeny of experimental crosses between these populations. There was no evidence of contemporary recombination between the maternal and paternal mtDNA. However, evidence for paternal leakage was observed in the experimental crosses of the two most divergent populations. In the progeny of these crosses, population-specific primers amplified both maternal and paternal mtDNA, with the paternal mtDNA dominant in several progeny. Therefore, even in a system with maternal and paternal mtDNA present, and that is capable of recombination, contemporary recombination was not detected. These results suggest that, under appropriate conditions, mtDNA paternal leakage and recombination are readily detected, but that recombination does not necessarily occur following paternal leakage. The use of G. pallida as a model organism for studying mtDNA mechanisms also has practical applications. Cyst-forming nematodes are economically important agricultural pests. Identifying suitable molecular markers for these nematodes, and understanding the evolution of these markers, will assist in developing effective prevention and treatment strategies. The ability of three molecular markers to resolve multiple representatives of five G. pallida populations was assessed. Targeted DNA regions included the ~3.4 kb scmtDNA region described above, a pathogenicity factor – the rbp-1 gene, and the internal transcribed spacer (ITS) region. Neighbour-Joining and Bayesian Inference methods of phylogenetic analysis were performed on the three DNA regions separately, and on a data set of these three regions combined. The phylogenies of the scmtDNA region and the combined data set resolved more populations as reciprocally monophyletic than did the phylogenies of the ITS region and rbp-1 gene. These results suggest that individual markers, particularly the ITS region and the rbp-1 gene, may be inadequate for distinguishing populations of G. pallida. The use of this scmtDNA marker may provide further insights into the historical distribution of G. pallida. The scmtDNA molecules of Globodera species have another unusual feature; polythymidine [poly(T)] length variation. Poly(T) variation can introduce frameshift mutations that may render genes as non-functional pseudogenes. Thus it is necessary to correctly characterise poly(T) tracts, as artefactual poly(T) variation could result in inaccurate phylogenetic inference or genome annotation. The extent of artefactual poly(T) variation was assessed using a cloned molecule from the cyst-forming nematode Heterodera cajani. This indicated that artefactual poly(T) variation rates were not significantly different to poly(T) variation rates that were measured in a biological sample after an amplification. This suggested that the majority of poly(T) variation in the biological sample was artefactual. The generation of poly(T) variation in a range of templates with tracts up to 16 Ts long was also examined, utilising the mtDNA of the cyst-forming nematodes. This indicated that poly(T) variation was present at tracts with >6 Ts, T deletions were 5 times more frequent than insertions, and a trend towards increasing error rates with increasing tract length. These observations have implications for phylogenetic and genomic studies of cyst-forming nematode mtDNA, as the mtDNA of these nematodes have unusually high numbers of poly(T) tracts.

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Unless otherwise indicated, the views expressed in this thesis are those of the author and do not necessarily represent the views of the University of Wollongong.