Development and validation of five native Aotearoa New Zealand genera-specific large brown macroalgal eDNA ddPCR assays

Environmental DNA is a rapidly growing methodology for ecological studies, leading to an increase in innovative techniques, such as the droplet-digital PCR (ddPCR), that can quantify and target DNA at a high rate of sensitivity for eDNA studies. Large brown macroalgae, Phaeophyceae, are an ecologically significant, diverse, and abundant benthic group in temperate-reef coastal environments. Members of Phaeophyceae, such as Laminariales and Fucales, are becoming recognized as potential significant contributors to Blue Carbon, although measurement of their contribution is challenging. The goal of the present study was to track the fate of specific large, brown macroalgae (kelp) genera detritus, by designing and validating five assays targeting the eDNA of kelp genera that are abundant in the coastal environments of Aotearoa New Zealand: Carpophyllum, Cystophora, Ecklonia, Lessonia, and Macrocystis. Short, diagnostic sequences of the mitochondrial cytochrome c oxidase subunit III gene (cox3) were targeted using ddPCR analysis. Specificity and sensitivity testing were conducted to validate the design of each assay for future use. All five assays produced no cross-amplification and had limits of detection (LoD) that were less than 1 copy/{micro}l. These novel, highly sensitive assays will be beneficial for future experimental and in situ eDNA studies that focus on any of these indigenous genera.