From sequence to validation, finding the genetic basis of a sexing trait in Bactrocera cucurbitae

The melon fly Bactrocera cucurbitae (Coquillette), is a destructive agricultural pest and is among one of the many species that is the target of expensive population eradication and suppression efforts within state and federal... [ view full abstract ]

The melon fly Bactrocera cucurbitae (Coquillette), is a destructive agricultural pest and is among one of the many species that is the target of expensive population eradication and suppression efforts within state and federal departments of agriculture. Area-wide integrated pest management programs control some species such as the Mediterranean fruit fly and the Mexican fruit fly through the release of sterile males known as the sterile insect technique (SIT). This method is not currently employed for B. cucurbitae in the United States though a genetic sexing strain (GSS) with a sexually dimorphic pupal color morph that can be used for mass-rearing and release does exist for the melon fly. The purpose of this project was to identify the causative gene and mutation of the genetic sexing trait white pupae (wp) in the genetic sexing strain of B. cucurbitae. To do so, we leverage the availability of a complete and highly contiguous reference genome and combine QTL mapping, 3’ mRNA sequencing, and low-coverage whole genome re-sequencing to investigate the possible causative mutation for white pupae and genes that are involved in the pupal color process. These methods combined allowed for extensive sampling and replicates to improve statistical power at a substantial reduction in cost. To validate the function of one of the genes identified through differential expression analysis and whole-genome re-sequencing, we designed single guide RNAs targeting PAM sites on one exon and injected a total of 1500 embryos. Upon emergence, injection survivors will be backcrossed in isolated matings to flies of the opposite sex from the same line. G1 Offspring of these G0 matings will be intercrossed to produce G2 individuals where the wp phenotype will be observed if the gene confers the trait of interest. Ultimately, the results of this study can be used to develop novel genetic sexing strains for use in SIT of other economically important agricultural pests.