Ragweed Species Identification by tRFLP of Pollen Grains.

Microscopy is the traditional method of identifying pollen grains in forensic palynology; the grains are identified by the morphology of the exine. Microscopy however does present some problems when identifying pollen grains to species level.

TRFLP is a technique that is used in molecular biology to differentiate between individual species in mixed communities. The method works by using PCR to amplify an area of the DNA, restriction enzymes are then used to digest the amplified products at a specific restriction. The restriction site varies depending on the enzyme being used. The TRFLP data can then be used to identify individual species as well as being used as a fingerprint[i] of the different assemblage of species present in the sample being analysed. TRFLP has been used to identify different species of both bacteria[ii] and fungi[iii].

The gene used for this study was the alcohol dehydrogenase 1 (Adh1). A previous attempt to develop a TRFLP based method of identification for pollen grains was made[iv], however there has been limited work to develop or expand the method further to allow for widespread use. TRFLP was used to attempt to differentiate between six different species of ragweed pollen. The restriction enzyme used was Msp1.

Statistical analysis of the data produced showed that the different species could be differentiated using TRFLP analysis and that the technique produced reproducible results. These results provide a novel development in the field of molecular palynology, which in turn could have a significant impact on the scope and implementation of palynology in a forensic setting. 

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[i] Thies J. E. (2007) “Soil Microbial Community Analysis using Terminal Restriction Fragment Length Polymorphisms.” Soil Science Society of America Journal 71: 579-591.

[ii] Waldron, L. S., B. C. Ferrari, et al. (2009). “Terminal Restriction Fragment Length Polymorphism for Identification of Cryptosporidium Species in Human Faeces” Applied and Environmental Microbiology 75(1): 108–112.

[iii] Groenewald M., D. U. Bellstedt et al. (2000). “A PCR-based method for the detection of Phaeononiella chlamydospora in grapevines.” South African Journal of Science 96: 43–46.

[iv] Eliet J. R. and S.A. Harbison (2006). “The development of a DNA analysis system for pollen.” International Congress Series 1288: 825–827