Species identification is necessary to prevent introductions of exotic plant pests through global trade. Many of these pests are understudied and lack publicly available DNA sequence data on which rapid molecular identification methods can be based. One such lineage is the genus Chrysodeixis, which includes three species of potential concern for United States trade initiatives: C. includens, C. chalcites, and C. eriosoma. Here we describe a method to generate robust 45S rDNA profiles using long read sequencing in order to clarify evolutionary relationships and develop a real-time PCR identification technique. Such an identification tool will be useful in rapidly differentiating between Chrysodeixis species of quarantine concern where traditional morphological identification methods are insufficient. Molecular methods such as this greatly reduce the time spent identifying each specimen, allow for detection of eDNA, vastly increase throughput, and increase the probability of detection. The methods presented here will be generally adaptable to any understudied lepidopteran taxa that necessitates a molecular diagnostic assay and, with adjustment or testing of the primers, could be applied to any group for which development of rDNA profiles in a benchtop setting would prove useful.
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