Non-destructive DNA Analysis of Single Pollen Grains

Abstract Forensic palynology has played a crucial role in many criminal investigations, from burglary to homicide, rape, war crimes, terrorism, and drug trafficking. With the emergence of DNA barcoding, pollen genomics has immense potential to classify and identify pollen taxonomically from its genetic signature. In forensic analysis, a non-destructive method for evidence is essential. Thus, several challenges exist for the extraction of DNA from the pollen grains, such as keeping the grain intact for secondary analysis and being able to bypass the issue of DNA mixtures more likely to be observed in a pollen assemblage. In this study, a new method was developed to address these challenges and was designed to extract genetic material while not destroying the pollen grain on a single grain level. We demonstrate the efficiency of the method with Pinus echinata, Taxodium distichum, and Plantago lanceolata single pollen grains. Quantitative PCR amplification of two genetic markers ribulose bisphosphate carboxylase (rbcL) and internal transcribed spacer 2 (ITS2), and digital microscopy demonstrates the non-destructive nature of this novel DNA extraction method from single pollen grains.