Universal Mitochondrial Multi-Locus Sequence Analysis (mtMLSA) to Characterise Populations of Unanticipated Plant Pest Biosecurity Detections

Abstract

Biosecurity responses to post-border exotic pest detections are more effective with knowledge of where the species may have originated from or if recurrent detections are connected. Population genetic markers for this are typically species-specific and not available in advance for any but the highest risk species, leaving other less anticipated species difficult to assess at the time. Here, new degenerate PCR primer sets are designed for within the Lepidoptera and Diptera for the 3′ COI, ND3, ND6, and 3′ plus 5′ 16S gene regions. These are shown to be universal at the ordinal level amongst species of 14 and 15 families across 10 and 11 dipteran and lepidopteran superfamilies, respectively. Sequencing the ND3 amplicons as an example of all the loci confirmed detection of population-level variation. This supported finding multiple population haplotypes from the publicly available sequences. Concatenation of the sequences also confirmed that higher population resolution is achieved than for the individual genes. Although as-yet untested in a biosecurity situation, this method is a relatively simple, off-the-shelf means to characterise populations. This makes a proactive contribution to the toolbox of quarantine agencies at the time of detection without the need for unprepared species-specific research and development.