Population Genomics, Diagnostic Strain Determination, and SIT Characterization for the Mexican Fruit Fly Anastrepha Ludens

Title Population genomics, diagnostic strain determination, and SIT characterization for the Mexican fruit fly Anastrepha ludens Abstract The following is a proposal for the Plant Protection Act 2019. The primary purpose of this agreement is to develop population genomic resources and diagnostic tools for SIT strain determination and colony characterization for the Mexican fruit fly Anastrepha ludens. The Mexican fruit fly is a destructive agricultural pest across its range of Central America and Mexico. It poses a significant threat to U.S. agriculture due to its proximity to citrus production in south Texas and California. Prior work using DNA data sets has demonstrated that populations of the species can be distinguished using genome-wide double-digest Restriction Site Associated sequencing (ddRAD) markers but that increased sample sizes of lab colonies and wild populations are needed to enhance confidence in the technologies (Dupuis et al. 2019 EvolApp 12:1641-1660). In year one of this project, PPA funded work to expand data sets with additional fly collections using ddRAD methods and applied these methods to field collected flies from California and Texas and lab colonies from Black Pupal Strains and Willacy strain in Texas, and this work is underway. In year two of the project, additional insect colony collections will be processed using Whole Genome “shotgun” Sequencing (WGS) to 1) select additional SNP markers from larger datasets to support rapid identification of colony source and examine effects of hybridization between fly colony and wild flies, and 2) evaluate the efficacy of these WGS approaches for diagnostic marker development. In addition, year two will include technology development studies to convert SNP genotyping technology that was developed from ddRAD/WGS data into a multiplex amplicon panel that can be sequenced on an in-house Illumina iSeq platform. This technology can be integrated with other marker systems used to diagnose species to provide a single protocol for Mexican fruit fly diagnosis. This one-year suggestion supports the FY21 Plant Protection Act Implementation Plan by developing and improving identification and diagnostic capacity for high priority pests. The team will generate and examine genomic data sets to select informative markers and provide APHIS-PPQ with a bioinformatics pipeline for efficient insect source identification. The new WGS data from lab colonies will be used to develop faster single tube tests for testing source of BPS or Willacy flies. The impact of the work will be new resources for three purposes: 1) SIT strain determination for the A. ludens rearing lines used for SIT in the U.S. and abroad; 2) genetic characterization of SIT strains used for mass rearing (assessing inbreeding, genetic diversity, heterozygosity, etc.), and 3) improved geographic source determination for trapped wild flies, focusing on northern and eastern Mexico adjacent to the U.S. border. Our research team has extensive expertise in tephritid population genetics and developing molecular diagnostic tools (e.g. Dupuis et al. 2018 Biol Inv 20:1211-1228), which includes the first range-wide population genomic assessment of A. ludens (Dupuis et al. 2019 Evol App 12:1641-1660). With extensive collaborations across the tephritid research community, this project has a high likelihood of success. Specific objectives: The goal of this agreement is to develop and improve molecular identification and diagnostic capacity for Anastrepha ludens. We have two specific objectives, all of which expand on our continued development of large population genomic datasets as their foundation. Our objectives are: 1) Increase the genomic resolution of current population genomic datasets by sequencing a subset of previously sequenced (for ddRAD data) specimens with a whole genome shotgun (WGS) approach (to create marker sets 3-4 magnitudes larger than previously developed ddRAD datasets; i.e. millions of markers compared to a few thousand). 2) Develop a sequencing-based diagnostic genetic panel for SIT strain delimitation, SIT characterization, and geographic pathway analysis by converting informative markers from these ddRAD/WGS datasets into a multiplex amplicon panel that can be sequenced on the Illumina iSeq platform. Keywords: population genomics, Diptera, Tephritidae, Anastrepha, recurrently invading pests, molecular diagnostics, pathway analysis