Community Dynamics of Freshwater Picocyanobacteria and Development and Application of HIP 1 PCR (Cyanobacterial Typing Technique).

Harper, Katie Johanna (2005) Community Dynamics of Freshwater Picocyanobacteria and Development and Application of HIP 1 PCR (Cyanobacterial Typing Technique). PhD thesis, Lancaster University.

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Abstract

The main aim of this study was to further our understanding of the diversity of a freshwater picocyanobacterial community. The HIP 1 PCR typing technique was developed and applied to 506 isolates of picocyanobacteria from a field study on Esthwaite Water in 2000. This study has demonstrated that genetic diversity existed within the picocyanobacteria characterised morphologically by rod shaped cells and high phycocyanin pigment content. Picocyanobacteria of this morphology represented a significant proportion of the picocyanobacterial community within the lake in 2000, which was typical of summer conditions for that lake. Twenty-one HIP 1 types were defined; ninety isolates were not assigned to a type as they generated HIP 1 PCR products which were insufficiently similar to less than five other isolates. The diversity of HIP 1 PCR products generated from isolates within the types ranged from homogeneous complex patterns to similarity of one key PCR product and heterogeneity of minor products. It is likely that on further isolation and testing of isolates within the more heterogeneous types further splitting of these types will be appropriate. The HIP 1 types were each re-isolated on at least two sampling occasions (one month apart). Some types were isolated from sites spanning the sampling period demonstrating that there was some stability within the HIP 1 types within the picocyanobacterial community within Esthwaite Water over the summer of 2000. Some types appeared to be isolated more successfully at different times of the sampling period suggesting a temporal shift/changes of HIP 1 types in the picocyanobacterial community between June and October in Esthwaite Water. It was possible to demonstrate that three HIP 1 types were isolated at statistically different success rates from some spatial or temporal locations. It was concluded that picocyanobacteria are considerably more diverse than indicated by gross morphology. Picocyanobacteria of different genotypes may play a range of functional roles within the freshwater environment. The HIP 1 PCR typing technique may allow us to get a handle on the diversity of this group in order to investigate this further. Within this study the HIP 1 PCR typing technique was developed for application to field study. Several methods of DNA extraction were compared; the Dynabeads DNA Direct System 1 was selected for use in the field study as it produced the most reliable template for PCR at the lowest processing time and cost. The discrimination, specificity and reproducibility for the technique were investigated and the technique continued to show potential for application to field studies. A method was devised for assessing the similarity of PCR products amplified by HIP 1 PCR for application to the field study isolates. This method was demonstrated as sufficient to appropriately group the PCR products from four different templates of cyanobacterial DNA separated repeatedly on different electrophoresis gels. On application to the field isolates this method assisted with the grouping of isolates into HIP 1 types, however the final user interpretation of the PCR products was also necessary. It is also expected that on further isolation/analysis of these types further definition of HIP 1 genotypes will be appropriate. The HIP 1 PCR technique has been demonstrated to be a useful tool for assessing diversity within ecological studies. The major advantages of the technique compared to others are the discrimination, cost, speed and simplicity of the technique and capacity for analysis of large numbers of isolates. The major limitation of the technique is the requirement for isolation of cyanobacteria.