A Combined Molecular Analysis of Pollen Utilizing Lipid Profiling & DNA Barcoding for Plant Species Identification: A Forensic Application

Forensic palynology is a branch of botany which focuses on using pollen as trace evidence to link individuals or objects to a crime. It relies on the assumption that geographical regions have unique flora and pollen assemblages, allowing for investigators to pinpoint when and where a person has been. Despite the first reported use occurring in 1959, forensic palynology has been heavily underutilized since due to the tedious nature of traditional microscopic pollen analysis, a lack of trained palynologists, and the low evidentiary value of pollen in court.

To alleviate these issues, an orthogonal chemical analysis of pollen using lipid profiling and DNA barcoding strategies is presented here. Different plant species have unique chemical profiles which can be used like fingerprints for identification purposes. A single non-destructive extraction is performed to isolate pollen coat lipids and DNA contained within the grain. Lipid extracts are analyzed using liquid chromatography-mass spectrometry and annotated using MS/MS databases. Three highly variable genetic markers, rbcL, matK, and ITS2, are targeted using species-specific primers and monitored using quantitative polymerase chain reaction (qPCR). A library containing the unique lipid profiles and primers for each species will be created and used as a reference for identification of unknown samples. Real pollen samples will be collected from the same plants over a two-year period to determine changes (if any) to the molecular profile. Lastly, the completed library and raw data will be accessible to interested parties using freely available online repositories.

This combined molecular approach aims to improve forensic pollen analysis by providing an objective, high-throughput, and rapid method for pollen identification. The techniques used can be easily integrated with instrumentation found in most crime labs, and the non-destructive nature of the extraction allows for complementary microscopic analysis of the grains. Additionally, the orthogonal approach greatly improves the evidential weighting of forensic pollen analysis by limiting errors that may arise when either technique is used independently. CA/NCF