Assessing mitochondrial DNA (CO1) barcodes for measuring diversity of Schizaphis spp. and abundance of Aploneura lentisci
Podmore, C. (2015). Assessing mitochondrial DNA (CO1) barcodes for measuring diversity of Schizaphis spp. and abundance of Aploneura lentisci (Thesis, Master of Science (Research) (MSc(Research))). University of Waikato, Hamilton, New Zealand. Retrieved from http://hdl.handle.net/10289/9886
Permanent Research Commons link: http://hdl.handle.net/10289/9886
We examined the diversity of mitochondrial DNA (CO1) sequences in the New Zealand native aphids with a particular focus on the genus Schizaphis (syn. Euschizaphis). Previously, the genus was thought to consist of two species, each host-specific to endemic New Zealand plants (Aciphylla and Dracophyllum). These unnamed native taxa are reasonably common but have either a narrow distribution (Aciphylla-host) or low local abundances (Dracophyllum-host). There is also some uncertainty over the number of Schizaphis species present on the various host plants. Specimens from both host plants were collected from ten sites in the North (n=3) and South (n=8) Islands. A total of 29 new COI sequences were obtained. A further 189 sequences were obtained from the Barcode of Life Datasystems (n=187) and GenBank (n=2). Thirteen of the 15 known native aphids were analysed by Maximum Likelihood and their taxonomic classification confirmed, the majority belonging to the Aphidinae, but also representatives in other sub-families. Maximum Likelihood analyses and pairwise genetic distances confirmed taxonomic groupings of the Aphididae sub-family Aphidinae and within the Aphidinae the monophyly of the sub-tribe Rhopalosiphina which contains two genera: Rhopalosiphum spp.and Schizaphis spp. Two distinct, well supported clades within Schizaphis were clearly delineated according to the host plant. However, we also found two distinct clusters within the Aciphylla-host South Island individuals and four within the Dracophyllum-host individuals (North & South Islands), suggesting six potentially cryptic species. Based on these data, we suggest that the CO1 gene region is effective for identifying aphids and could assist in the on-going discovery and protection of these taxa in New Zealand. New information regarding the diversity of New Zealand native Schizaphis aphids is using COI gene sequences to reveal additional diversity. International databases such as the Barcode of Life Datasystems and GenBank will be particularly helpful in examining global relationships within the New Zealand native aphid taxon. Root Aphids (Aploneura lentisci) are an introduced taxon, which live exclusively on the roots of Poaceae and can cause detrimental effects to New Zealand pastures, reducing the forage available for farmed livestock. In order to assess abundances of A. lentisci in soil samples, specific primers were first developed to target and amplify the mitochondrial gene region cytochrome c oxidase subunit 1 (CO1) gene region. Three DNA extraction protocols, the PowerSoil® DNA Isolation Kit , Extract-N-Amp™Tissue PCR and a phenol chloroform DNA extraction protocol were then assessed and real time quantitative polymerase chain reaction (qPCR) was used to measure the amount of amplicon produced, which was compared to a standard curve of known aphid numbers. The developed primers successfully amplified a 317 nucleotide fragment of the COI gene region and had limited cross reactivity with other aphid taxa tested. Of the three extraction methods, the Extract-N-Amp Tissue PCR Kit was the most effective. Adult root aphids were selected as representatives of infestations. DNA concentrations extracted using the Extract-N-Amp Kit were linear over the entire range of aphid numbers (R²=0.98). Further dilutions were carried out: 10-fold, 100-fold and 1000-fold to test linearity and when combined produced a standard curve with a regression of 0.93 over a cycle threshold range of 18-33 cycles. DNA extractions from mixtures of 60 aphids in soil produced amplifiable aphid DNA. We conclude that this method will successfully measure root aphid abundances from soil samples using mitochondrial CO1 gene sequences and qPCR.
University of Waikato
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