DNA metabarcoding is an important tool for molecular ecology and environmental management. However, its success depends on the availability of complete genetic reference databases and the taxonomic resolution of polymerase chain reaction (PCR) amplicon sequences used. Metabarcoding has the potential to revolutionise biodiversity monitoring by simplifying environmental sampling, providing reliable species identifications without a need for specialist taxonomic expertise, thereby reducing costs. However, the requisite mitochondrial reference libraries of DNA sequences are currently lacking for most taxa, and sequencing methodologies require further development. Here, we evaluate whether existing universal primer sets (i.e., MiFish, MiDeca and ecoPrimers) are good candidates for environmental DNA (eDNA) metabarcoding for 88 aquatic biota (vertebrate and invertebrate) in the Alligator Rivers Region and whether they produce amplicons in silico and in vitro with enough nucleotide distances to distinguish target species from one another. Further, we investigate whether it is possible to amplify the mitochondrial genomes (mitogenomes) of target species through mitochondrial enrichment primers to fill in the gaps in the reference database. I first performed a database review of available mitochondrial genes and mitogenomes in order to develop a reference library database for aquatic species occurring in the Alligator Rivers Region. Second, I identified suitable mitochondrial enrichment primers for fish and assessed them in silico against the database using the bioinformatics program Geneious. Finally, I assessed whether the universal primer sets MiFish, MiDeca and ecoPrimers are able to amplify and provide enough base pair difference in silico between target species for future biodiversity assessments. To this end, I demonstrate that two fish primers can amplify whole mitogenomes in vitro, and the universal primer sets MiFish, MiDeca and ecoPrimers (5 primer pairs) match nearly all of the target species in silico. Nearly all universal primer sets provided the most taxonomically discriminative barcodes; however, there is a tradeoff between all-inclusive universal primers and accuracy of taxonomic assignment. Furthermore, I amplified barcodes for 38 of 39 extracted DNA fish species using the MiFish primers. Together these results demonstrate the technical feasibility of using an eDNA metabarcoding approach to survey aquatic biota in the Alligator Rivers Region.
|Date of Award||2022|
|Supervisor||Sam Banks (Supervisor)|