Abstract
Seed shipments, silos and storage houses often contain weed seeds or seeds of restricted crops such as undeclared genetically modified (GM) varieties. Random sub-sampling is the favoured approach to detect unwanted biological materials in seed lots but is prohibitively expensive or else ineffective for the huge volumes of seeds moved in commercial operations. This study uses maize and cowpea seed admixtures as an exemplar to evaluate the feasibility of using aerosol sampling of “seed dust” as an alternative to seed sub-sampling. In an initial calibration phase, qPCR of the rbcL barcode followed by high-resolution melting (HRM) of a DNA titration series revealed a strong linear relationship between mix composition and HRM profiles. However, the relationship became skewed when flour mixes were used to build the titration, implying a DNA extraction bias favouring cowpea. Aerosol samples of seed dust above a titration of mixed seed samples were then collected along vertical and lateral axes. Aerosols were characterised by light microscopy, qPCR-HRM and next-generation DNA sequencing (Illumina MiSeq). Both molecular approaches again showed bias but this time in a reverse direction to flour samples. Microscopic examination of the aerosol sample suggested this divergence could be attributed to differences in abundance of airborne starch particles. Despite the bias, it was nevertheless possible to estimate relative abundance of each species using the abundance of minibarcodes. In light of these results we explore the feasibility of aerosol sampling for commercial seed lot characterisation.
Highlights
Most countries impose import restrictions to control the accidental or intentional introduction of seeds from unauthorised crops, invasive and noxious weeds, or more recently, from genetically modified (GM) seeds [1, 2]
Having established that high-resolution melting (HRM) melt curve analysis could be used to describe a DNA template titration series, the step was to establish whether the same approach could be used to reflect mixtures of flour samples made from ground grains of the two species
This is a necessary step as any differences in the efficiency of DNA extraction from grains of the two species would introduce divergence from the simple linear relationship observed above between DNA species mix and HRM profile
Summary
Most countries impose import restrictions to control the accidental or intentional introduction of seeds from unauthorised crops, invasive and noxious weeds, or more recently, from genetically modified (GM) seeds [1, 2]. Enforcement of these restrictions is made difficult by the sheer volume of seed involved in commercial operations and by the complexity of seed handling processes. Funding for most MJW time received from Biotechnology and Biological Sciences Research Council (BBS/E/0012843C) (https://bbsrc.ukri.org). The funder roles funded for conceptual aspects of data interpretation and analysis, and for writing
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