D3486: Evaluating diagnostic loci for use in Bactrocera dorsalis complex species identification

The Bactrocera dorsalis complex is a grouping of fruit fly species that appear similar morphologically to the global pest species Bactrocera dorsalis. This group contains over 89 species, with most species not related evolutionarily. As a result, many species can be identified using either DNA methods or expert examination of adult morphology. However, within the larger complex are 12 species that belong to a clade called the Bactrocera dorsalis group (or B. dorsalis s.l.). This clade includes several significant pests that the standard COI DNA barcode methods cannot identify due to introgression. The development of additional diagnostic markers or a multi-locus Sequencing Type (MSLT) approaches that analyze multiple fragments was proposed as a possible solution to provide accurate identifications. By comparing the genomes of B. dorsalis, B. correcta, and B. zonata, we identified 5658 single-copy orthologs among these species. Gene tree comparisons of these orthologs identified 15 diagnostic genes that could differentiate between species within the Bactrocera dorsalis group. Diagnostic gene sequences were split by exon, and primers were designed for the amplification and sequencing of these exons. We tested the utility of these primers using samples from known and unknown species. Gene tree analyses of each exon failed to diagnose species due to a lack of significant variation within a single exon. However, the concatenation of different combinations of exon sequence alignments (at least three exons) provided increased resolution and were able to identify all species within Bactrocera dorsalis group. These MSLT primers provide the first step in creating a fast and accurate molecular identification pipeline for this global pest.