Molecular characterisation of the commercially important Agathosma species
Thesis (MSc (Plant Biotechnology))--University of Stellenbosch, 2006.
The development of a reliable and reproducible method for the genetic characterisation and identification of the commercially important Agathosma species was investigated. Previous research attempts aimed at developing a reliable and reproducible method of identifying these Agathosma species failed, mostly because these studies were based on phenotypic traits and these methods were therefore influenced by environmental factors. In this study amplified fragment length polymorphisms (AFLPs) were successfully used to quantify the genetic variation between the Agathosma species and as a result three distinct groups could be identified. The data obtained were elaborated with the Dice genetic similarity coefficient, and analysed using different clustering methods and Principle Coordinate Analysis (PCoA). Cluster analysis of the genotypes revealed an overall genetic similarity between the populations of between 0.85 and 0.99. The AFLP-based dendrogram divided the populations into three major groups: (1) the A. serratifolia and A. crenulata populations, (2) the putative hybrid, A. betulina X A crenulata populations, and (3) the A. betulina populations, confirming that this technique can be used to identify species. The question of hybridisation was also clarified by the results of the PCoA, confirming that the putative hybrid is not genetically intermediately spread between the A. crenulata and A. betulina populations, and that it is genetically very similar to A. betulina. The putative hybrid can therefore rather be viewed as a genetically distinct ecological variant of A. betulina. As the AFLP technique cannot be directly applied in large-scale, routine investigations due to its high cost and complicated technology, the development of polymerase chain reaction (PCR)-based molecular markers, able to accurately identify the species, was undertaken. Due to the superior quality of A. betulina oil, the development of such markers is especially critical for this species. Several species-specific AFLP markers were identified, converted to sequence characterised amplified regions (SCARs) and ultimately single nucleotide polymorphisms (SNPs) were characterised. The developed SCARs were unable to distinguish between the species. The conversion of AFLP fragments to SCARs is problematic due to multiple fragments being amplified with the AFLP fragment of interest. The diagnostic feature of the SNP-based markers was not sensitive enough, since this technique could not distinguish between the A. betulina and A. crenulata and/or the putative hybrid populations. The SNPs that were characterised were found not to be species-specific; they were only specific to the particular clone. Although a quick and robust marker specific for A. betulina has not yet been developed, this study sets the stage for future genetic studies on Agathosma species. Such a marker, or set of markers, would be an invaluable contribution to a blooming buchu oil industry.