Abstract:
The purpose of this research was to develop DNA based techniques for the analysis of botanical forensic evidence. The research included the analysis of Cannabis, the investigation of illegal logging and the analysis of plant material as trace evidence. A set of single nucleotide polymorphisms (SNPs) within the tetrahydrocannabinolic acid synthase gene, corresponding to functional and non-functional forms of the enzyme, were assayed using a mini-sequencing approach (SNaPshot). This enabled the differentiation of Cannabis containing high levels of the psychoactive agent tetrahydrocannabinol (THC) from low THC Cannabis. A multiplex of eleven STR loci was developed and tested across a set of 82 New Zealand Cannabis samples. Typing of New Zealand Cannabis revealed significant departure from Hardy-Weinberg equilibrium, likely due to the effect of clonal reproduction on genotypic diversity in the New Zealand Cannabis population. Unusual triallelic patterns were observed at some loci in a small number of individuals. Sequencing of homozygotes revealed that while most alleles contained the expected repeat region point mutations and insertion/deletion events were also present in some alleles. In one notable example the published repeat sequence did not appear to vary. A set of 13 Inter-simple sequence repeat (ISSR) primers were amplified in the native forest tree Rimu (Dacrydium cupressinum). Although the ISSR primers tested were variable the length of the amplified fragments was considered unsuitable for the analysis of Rimu in the context of illegal logging. A set of primers were designed to target taxon specific fragments of the internal transcribed spacer (ITS) of the nuclear ribosomal DNA (nrDNA). Amplification of taxon specific ITS fragments from experimentally derived mixed samples was successful, however difficulties were encountered with the differentiation of similarly sized amplicons with the microfluidics platform chosen. Of the three aspects of DNA analysis forensic botanical material covered in this thesis the DNA analysis of Cannabis material shows the greatest promise. Although the techniques developed for the analysis of plant fragments from mixed samples and the analysis of Rimu in the context of illegal logging were less successful there is great potential for the use of DNA technologies for the analysis of these evidence types as well.