Location: Tropical Pest Genetics and Molecular Biology Research Unit
Project Number: 2040-22430-028-003-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Aug 1, 2019
End Date: Sep 30, 2022
The major objectives of this three year project are to: 1. Screen the genomes of existing medfly genetic sexing strains to characterize the mutations causing the temperature sensitive lethal and white pupae traits. 2. Characterize the exact translocation breakpoint between the Y chromosome and the 5th autosome in the males of the Veinna-8 strain that confers these traits to be female specific. 3. Adapt CRISPR-based methods to recreate the translocation in wild type lines. 4. Demonstrate a targeted gene editing approach to re-creating temperature sensitive lethal in Anastrepha and Bactrocera species.
Two genome sequencing approaches will be used for characterization of the mutations conferring white pupae and temperature sensitive lethal. Through cataloging SNPs that are in common to each line, at expected frequency in each sex surrounding the QTL locus, a short list of putative causative mutations can be generated. These putative mutations will be screened using CRISPR/Cas9 targeted gene editing to validate, and additionally targeted in new tephritid species. Additionally, several distinct genomic methodologies will be employed to characterize the translocations present in the medfly genetic sexing systems. Four distinct chromosomal arrangements will be investigated, a wild type, a Vienna-7 line, and a Vienna-8 with or without D53 inversion (i.e. V8-D53+ and V8-D53-). For each, a single male file (containing the autosomal/Y translocation) will be prepared for one of three methods, optical mapping, a HiC contact map construction, and linked-read sequencing.