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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 #444148

Research Project: Integrative Identification Methods for Bactrocera Fruit Flies (Year 3)

Location: Tropical Pest Genetics and Molecular Biology Research Unit

Project Number: 2040-22430-028-025-I
Project Type: Interagency Reimbursable Agreement

Start Date: May 1, 2023
End Date: Dec 31, 2024

The expected outcome of the agreement is to develop a set of diagnostic markers for species identification of the Queensland fruit fly (B. tryoni). The objective for this goal is to seek and identify a set of informative molecular markers for species identification based on regions with high genetic differentiation that will be tested using a phylogenomic approach.

Bactrocera tryoni species complex whole genome resequencing (WGS) and species delimitation. Single insect whole genome sequencing will be performed for a series of B. tryoni species complex specimens, utilizing Illumina-based sequencing methods. We will generate at least 100 new DNA extracts of representative specimens from different regions of the B. tryoni species complex that were obtained and are available in the University of Hawaii Insect Museum. We will perform paired-end 150 bp sequencing to reach at least 20X coverage for each specimen to replicate samples from unique geographic and morphological types for this complex. No current reference genome exists for all species in this complex, so a contig level reference genome will be generated from a single adult fly using PacBio HiFi sequencing. One SMRTcell 8M run will be performed from a low-input SMRTbell Express 2.0 library preparation, resulting in ~40X coverage of the genome. The genome will be assembled using HiFiASM to contig level, duplication will be removed using purge_dups to generate a reference genome for the short-read sequencing to be mapped. The short reads will be mapped using bwa, processed for single nucleotide polymorphisms (SNPs) using Deepvariant + GLNexus and filtered using vcftools. Resulting SNPs will be used in a phylogenomic approach to infer the phylogenetic relationship between members of the complex and delimitate the species/lineages, which may be required for specific pest management strategies. Then, we will calculate genetic differentiation (Fst) across the genome and select regions with higher Fst values to be employed as set of diagnostic markers. The phylogenetic relationships obtained from the set of markers will be compared with the phylogeny obtained from the complete dataset. We seek that the phylogenetic information of this reduced group of SNPs to be equivalent to that found at genomic level. We will additionally sequence COI of approximately 1,000 specimens (including WGS specimens) using the PacBio platform to test the efficacy of this DNA barcode marker to identify B. tryoni species complex and diagnose pests. The genome assembly and raw WGS data will be accessible at GenBank database and available to APHIS prior to submission upon request.