Preliminary application of DNA barcoding toward the detection of viable plant propagules at an initial, international point-of-entry in Georgia, USA
Over 90% of global commercial trade occurs between seaports, which are initial points-ofentry for nonnative, potentially invasive propagules. As such, there is a need to develop means to both rapidly intercept and identify propagules as they arrive. Here, we focus on plant propagules that are assumed to be non-native, in seed form. Because standardmorphologicaltechniquesalonearelaborious and require taxonomic expertise, we sought to address if identiﬁcation through barcoding of the plastid DNA based on genetic analysis? (4) Are nonnative invasive plant species present on shipping containers entering the Port of Savannah? This research collected 11,044 seeds from 628 refrigerated shipping containers between 2015 and 2017. Seeds were then morphologically sorted into Seed Types. Barcoding of the matK and rbcL gene regions of the plastid genomes directly isolated from seeds resulted in poor ampliﬁcation. This is likely due to a host of potential confounding factors. Therefore, we germinated seeds and utilized leaf-tissues for sequencing of these two gene regions. From BLASTn analyses, results returned top hits for a variety of species, with up to 22 possible nonnative plant species and one deﬁnite Federal Noxious Weed. This work investigates the interception application of DNA barcoding to improve agro- and bio-security issues posed by nonnative and invasive species. Though this study required the germination of the seeds to obtain leaf-tissue suitable for our DNA barcoding method, we effectively demonstrated seed viability. Our seed identiﬁcation process was lengthy and understandably not feasible for real-time application. Therefore, we seek to improve our methods for future applications by testing other approaches that may better complement morphological identiﬁcation. Next reasonable steps include improved extraction protocols, metabarcoding to generate DNA barcode sequences directly from groups of seeds harvested from shipping containers and implementing other next-generation sequencing techniques.