Project Number: 3091-22000-038-67-I
Project Type: Interagency Reimbursable Agreement
Start Date: Oct 1, 2019
End Date: Sep 30, 2020
1) Assist in acquisition of weevil samples by identifying localities/sources for weevil collections, organizing collection trips, and obtaining appropriate permits. 2) Participate in the application of hyRAD-MYbait on samples with low quality DNA and sequencing of additional samples. 3) Participate as one of three labs to identify informative SNPs and develop diagnostic assays, then refine and validate a diagnostic assay to distinguish Anthonomus grandis subspecies and populations.
Species identity of all fresh samples collected from the field or preserved specimens from museum collections will be verified morphologically. Given the small size of weevils and relatively low yield of genomic DNA, whole individuals will need to be destructively sampled to obtain sufficient DNA for downstream analysis. Replicate insects for each taxonomic sample will therefore be assigned to one of three groups: (1) representative male and female voucher specimens will be retained for subsequent morphological taxonomic reference, (2) half of the remaining insects will be available for use in initial SNP marker and diagnostic assay development, and (3) the remaining half of the insects will be stored for later use as samples of known origin in the diagnostic assay validation phase of the project. We will use our existing boll weevil ddRADseq data as the basis to implement a recently-developed approach called hybridization RAD (hyRAD) that will enable us to collect DNA sequence data from across the genomes of a range of weevil species that will include Anthonomus grandis grandis, A. g. thurberi, A. texanus, A. fulvus, A. hunteri, A. cognatus, A. peninsularis, A. eugenii, A. townsendi, A. palmeri, and Conotrachelus nenuphar. This data will be used to discover SNP markers for development of a rapid molecular diagnostic assays that can provide accurate taxonomic determinations. Using the SNP data collected by ddRAD-seq, we will design custom TaqMan SNP Genotyping Assays to identify the target weevil taxa. These new diagnostic tools will utilize established quantitative PCR protocols that can be implemented across laboratories. This new identification capacity will: 1) provide faster, more accurate determinations; 2) improve the diagnostic capacity for APHIS and other action agencies, and therefore improve program decisions; and 3) identify potential pathways for entry/reinfestations to help mitigate these events.