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ARS Home » Pacific West Area » Hilo, Hawaii » Daniel K. Inouye U.S. Pacific Basin Agricultural Research Center » Tropical Pest Genetics and Molecular Biology Research Unit » Research » Research Project #443228

Research Project: 22-8130-0984 Molecular Diagnostics for the Mango Fruit Fly Complex Year 2

Location: Tropical Pest Genetics and Molecular Biology Research Unit

Project Number: 2040-22430-028-023-S
Project Type: Non-Assistance Cooperative Agreement

Start Date: Sep 30, 2022
End Date: Sep 29, 2023

The expected outcomes of the agreement are to (1) develop diagnostic markers for species identification and tracking to source island for the mango fruit fly (B. frauenfeldi) based on a phylogenomic approach and (2) test the molecular markers for this complex.

1. Genome reference construction A de novo assembly will be generated for a representative sample within the frauenfeldi complex to serve as a reference to use for mapping whole genome sequencing reads from the specimen selected below. To generate the reference assembly, a single specimen will be selected. Ideally, a fresh collected specimen will be used, field homogenized in extraction buffer and directly shipped for DNA extraction and library prep. If fresh material is not available, a previously collected sample will be used (stored frozen in ethanol). Previously collected specimens may be of lower quality and yield than fresh specimens, but either should be sufficient for the study. 2. Diagnostic markers for the mango fruit fly (B. frauenfeldi) species complex. Cooperator will work on selecting a set of about 40 specimens that adequately reflects the species complex and geographic variation. Cooperator will use the methods that we have successfully developed for diagnosis markers of Anastrepha fraterculus complex based on whole genome data. This method consists in comparing the topology of the phylogenies inferred from independent loci alignments and a phylogeny constructed from all evaluated loci (species tree). It is expected that phylogenies of highly informative loci show the same or very similar clade formation as in the species tree. Cooperator will check if molecular markers already developed for species identification of other Bactrocera species by Cooperator are in the informative loci list generated for B. frauenfeldi. We will seek to select those markers over other loci with the intention, as far as possible, of unifying the diagnostic markers for the genus. 3. Testing markers for B. frauenfeldi complex We will develop primers for an estimated about 20 target loci in the complex, which should provide similar species and/or population identification profile as the complete genome data for the samples we have this information available. We will then test performance of primer sets by amplifying and sequencing using high throughput sequencing across approximately 200 specimens. The effectiveness of the diagnostic molecular markers will be evaluated based on reliable amplification and sequencing of flies and quality and informativeness of sequence data.