Chloroplast Markers for Detecting Chinese Tallow (Triadica sebifera) DNA in Environmental Samples

DNA analysis of environmental samples (eDNA) provides a non-intrusive approach to identify organisms, characterize biological communities, and assess biodiversity, including the detection and monitoring of invasive plant effects. However, the use of eDNA for specific applications, such as targeted-species detection, geographic and floral source tracing, and assessment of invasive plant ecological and environmental effects, requires the development of species-specific genetic primers. Chinese tallow (Triadica sebifera (L.) Small) is a non-native high-impact invader, capable of changing fire regimes, native biodiversity, nutrient cycling, and wildlife habitat and populations, that is expanding in range and abundance throughout the southern United States. In this study, we investigated and identified specific genetic sites, markers, in the tallow chloroplast genome and developed sets of primers for tallow eDNA detection. Two sets of tallow primers were developed, tallow-specific primers and tallow-related primers. Both sets of primers can be used for tallow eDNA detection, with higher target specificity for tallow-specific primers. Primers were subsequently validated for target specificity against closely related species, samples of tallow tissue, and honey and honey bee-collected pollen from areas with tallow. We found that tallow-specific primers differentiated tallow eDNA from closely related species, demonstrating target specificity. Furthermore, a sequence analysis of the tallow-related primers in the polymerase chain reaction accurately distinguished members of the Hippomaninae subtribe, including tallow, from other subtribe or subfamily members within the Euphorbiaceae. Ultimately, the genetic markers and the corresponding sets of primers will facilitate eDNA analysis of tallow for several applications, including detection and monitoring in water and soil, assurance of honey quality and floral source tracing, and perhaps serving as a model for determining plant use by pollinators.