DNA-barcoding the rusts – sampling herbarium specimens in the National Mycological Herbarium (DAOM).

Plant pathologists, diagnosticians and regulatory agencies rely on access to comprehensive, accurate, and curated reference DNA sequence databases. These data are essential for rapid and authoritative identifications of plant pathogens in field samples, commodity imports/exports and environmental samples as represented by DNA sequences derived from next-generation sequencing (NGS) techniques. Herbarium collections are the most easily accessible source of obligate fungal pathogens and their hosts, with specimens often dating back to the 1800s and representing a broad diversity of hosts and geographic provenance. A processing pipeline was developed to minimize destructive sampling of these irreplaceable collections while maximizing DNA yield and facilitating high throughput. DNA-barcoding techniques were applied to both pathogen and host, targeting the ITS2 and rbcLa gene regions respectively, for over 1200 specimens of rust fungi (Pucciniales/Uredinales) preserved in DAOM. For specimen selection, from the more than 38K available, we focused on species of quarantine concern as listed by various countries, those not yet represented in public sequence databases, those occurring on agricultural hosts, and alternate hosts for species with complex life cycles. Our success rate ranged from 51-72% (rusts, depending on genus) and 60% (hosts), and the oldest specimen successfully sequenced for both was collected in 1889. We now have consensus sequences representing 88% (23/26) of genera and 73% (242/330) of species sampled. The primary impacts will be to increase the number of publicly available and authoritatively identified reference DNA barcodes and reduce the risk of misinterpreting DNA signatures of innocuous species with those of regulated species.