A New Diagnostic Resource for Ceratitis capitata Strain Identification Based on QTL Mapping

Abstract

The Mediterranean fruit fly Ceratitis capitata (Wiedemann) is a destructive agricultural pest and the subject of exclusion efforts in many countries. Suppression and eradication of invasive populations to prevent its establishment is facilitated by the release of sterile males using the sterile insect technique (SIT). In SIT release areas, it is critical to accurately discriminate between released sterile males and wild individuals to detect extremely rare invasive individuals in areas inundated with millions of sterile male flies. Current methods for discrimination exist but are not always definitive, and a more reliable method is necessary. To address this, we developed a genotyping assay that can be used to discriminate between sterile males from the SIT strain and wild individuals. This was achieved by identifying single nucleotide polymorphisms (SNPs) linked to the maintained traits that facilitate male-only releases, white pupae (wp) and temperature-sensitive lethal (tsl), via QTL mapping. This resulted in the identification of one SNP that was in near-perfect linkage disequilibrium between genotype at this locus and the pupal color phenotype. Medfly from many SIT colonies and wild individuals from across its geographic range were genotyped for this locus, and results show its consistency in identifying SIT flies. In addition, linkage and QTL mapping of wp and tsl have larger impacts as they can serve as foundational tools to identify the genetic basis of traits that facilitate the separation of males from female flies, which can be used to develop SIT programs in related species.

Keywords: QTL; diagnostics; genome-wide genotyping; linkage mapping; medfly; sterile insect technique.

Figure 1

(A) Schematic of the sex chromosomes and the fifth chromosome containing the wp and tsl gene for female and male Vienna-8 GSS (genetic sexing strain) and HiMed C. capitata. The location of the white pupae and temperature-sensitive lethal gene has been crudely mapped to the tip of the fifth chromosome, and a translocation between the Y-chromosome and the fifth chromosome harbors a wild-type allele (wp⁺) in males and is heterozygous at the locus, in contrast to females who are homozygous for the wp and tsl mutations. (B) Drawing of the sex chromosomes and the fifth chromosome containing the genes for female and male wild-type C. capitata white pupae and wild-type temperature-sensitive lethal; wild-type individuals lack the wp and tsl variants. This figure is adapted from Franz (2005).

Figure 2

Virgin adult females from the C. capitata genetic sexing strain (Vienna-8 D53-) were mated in isolation with males from the wild-type laboratory colony (HiMed). The white pupae trait (wp) is autosomal recessive, so resulting F1 progeny will all have a wild-type brown pupal color phenotype. In F2 progeny from isolated intercrossing between F1 full-siblings, the pupal color phenotype will segregate at a 3:1 ratio of wild-type brown pupae to white pupae. White pupae F2 females were back-crossed to wild-type laboratory colony males. This increases the proportion of the wild-type alleles genome in subsequent offspring. Like the F1 progeny, the F3 progeny will all have a wild-type brown pupal color phenotype and full-sibs will be intercrossed to produce an F4 mapping population comprised of female and male wild-type brown pupae and white pupae individuals. Colors of the text boxes denote pupal color of individuals, white boxes represent white pupae individuals, and yellow boxes represent brown pupae individuals.

Figure 3

A discriminant analysis of principle components was performed on a total of 143 individuals from various sources using their genotypes for 77,844 single nucleotide polymorphism loci. All individuals have a membership probability of < 99% to one of seven clusters, Vienna strain flies were assigned to three clusters (red), and wild individuals formed four distinct clusters (yellow, green, violet, and blue). Individuals from Spain (diamonds) are denoted as some were assigned to the same cluster as Vienna strain flies (red diamonds), and some were assigned to the wild fly cluster consisting of flies originating from Australia, the Azores, Brazil, Hawaii, and Morocco (yellow diamonds). Individuals from California (triangles) are denoted as one was assigned to the wild fly cluster consisting of flies originating from Australia, the Azores, Brazil, Hawaii, and Morocco (yellow triangle), while others were assigned to the cluster containing individuals from Central America (violet traingles). The box denoting eigenvalues shows the relative variance described by each of the principle components in descending order and that the clusters can be differentiated using principle components 1 and 2 (x-axis and y-axis, respectively), which together explains 86% of the variance. The clusters roughly correspond to a geographic location, with flies collected from sub-Saharan African countries being the most distinct.

Figure 4

Quantitative trait loci analysis using the binary interval mapping model. Results indicate that the pupal color phenotype is tightly linked to loci on autosome 5. The dotted line represents a logarithm of the odds (LOD) threshold for significance as determined by a permutation test performed with 100,000 permutations (p < $1\text{e}-4$).

Figure 5

Weir and Cockerham’s Fst calculated between wild individuals and only Vienna colony females that are homozygous for wp using the 981 single nucleotide polymorphism (SNP) loci in the linkage map. The SNP locus identified through quantitative trait loci analysis as tightly linked to wp was colored red.

Figure 6

Allelic discrimination plot showing $\Delta$ Rn value for wild-type allele (T) on the x-axis vs. the $\Delta$ Rn value for the wp-linked allele (G) on the y-axis for individuals genotyped with assay AHMSY8D, where the $\Delta$ Rn value is the detected change between the initial read and the endpoint read in fluorescent signal of the reporter and the passive reference dye. Average values of three replicates are shown with minimum and maximum $\Delta$ Rn values for both alleles are shown as bars on their respective axes. Individuals that were assigned the genotype of homozygous for the wild-type allele are in orange, heterozygotes are in red, homozygous for the wp-linked allele are in blue, and the no template control (NTC) is in black.