A Chromosome-Scale Assembly of the Bactrocera cucurbitae Genome Provides Insight to the Genetic Basis of white pupae

Abstract

Genetic sexing strains (GSS) used in sterile insect technique (SIT) programs are textbook examples of how classical Mendelian genetics can be directly implemented in the management of agricultural insect pests. Although the foundation of traditionally developed GSS are single locus, autosomal recessive traits, their genetic basis are largely unknown. With the advent of modern genomic techniques, the genetic basis of sexing traits in GSS can now be further investigated. This study is the first of its kind to integrate traditional genetic techniques with emerging genomics to characterize a GSS using the tephritid fruit fly pest Bactrocera cucurbitae as a model. These techniques include whole-genome sequencing, the development of a mapping population and linkage map, and quantitative trait analysis. The experiment designed to map the genetic sexing trait in B. cucurbitae, white pupae (wp), also enabled the generation of a chromosome-scale genome assembly by integrating the linkage map with the assembly. Quantitative trait loci analysis revealed SNP loci near position 42 MB on chromosome 3 to be tightly linked to wp Gene annotation and synteny analysis show a near perfect relationship between chromosomes in B. cucurbitae and Muller elements A-E in Drosophila melanogaster This chromosome-scale genome assembly is complete, has high contiguity, was generated using a minimal input DNA, and will be used to further characterize the genetic mechanisms underlying wp Knowledge of the genetic basis of genetic sexing traits can be used to improve SIT in this species and expand it to other economically important Diptera.

Keywords: Diptera; Drosophila; Genetic sexing; Mendelian genetics; QTL; Sterile Insect Technique; Tephritid fruit flies; chromosome assembly; genomics; genotyping; linkage mapping; synteny; whole genome sequencing.

Figure 1

B. cucurbitae. (A) Front view of an adult female B. cucurbitae. (B) A male heterozygous at the wp locus featuring a wild-type brown pupal case (left) and a female homozygous for the wp allele featuring a white pupal case (right). Pupal color phenotype is the sexing trait used to sort males from females in the T1 Melon genetic sexing strain.

Figure 2

Sex chromosomes and autosome of genetic sexing strain and wild-type B. cucurbitae. (A) Drawing of the sex chromosomes and the autosome containing the wp gene for female and male T1 Melon B. cucurbitae. The location of the wp gene has been crudely mapped to the tip of one of the autosomes, and a translocation between the Y-chromosome and the autosome harbors a wild-type allele (wp⁺) in males and are heterozygous at the locus in contrast to females, who are homozygous for the wp mutation. (B) Drawing of the sex chromosomes and the autosome containing the gene for female and male wild-type B. cucurbitae; wild-type individuals lack the wp mutation. Figure adapted from Franz (2005).

Figure 3

Crossing scheme used to generate F4 mapping population. Virgin adult females from the B. cucurbitae white pupae genetic sexing strain were mated in isolation with males from the wild-type laboratory colony. The white pupae trait is autosomal recessive; resulting F1 progeny will all have a wild-type brown pupal color phenotype. In F2 progeny from isolated intercrossing between F1 full sibs, the pupal color phenotype will segregate at a 3:1 ratio of wild-type brown pupae to white pupae. White pupae F2 females were backcrossed 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.

Figure 4

Comparison of continuity in terms of N50 and proportion of complete BUSCOs in the gene set. B. cucurbitae (red circle) ranks high in continuity (x-axis) and proportion of complete BUSCOs (y-axis) compared to notable arthropods.

Figure 5

Linkage map. Genetic map depicting size of linkage group in centimorgan and distribution of SNPs in five linkage groups.

Figure 6

QTL map. QTL analysis using the binary interval mapping model. Results indicate that the pupal color phenotype is tightly linked to loci on the autosome chromosome 3. A permutation test performed with 100,000 permutations identified loci showing significant linkage to the phenotype (P < $1\text{e}-4$).

Figure 7

Superscaffolded map integrating linkage information from all chromosomes. For each linkage group the y-axis shows the position of each marker and its corresponding scaffold in the linkage map in centimorgans. On the x-axis is the subsequent position of each scaffold in megabase pairs after superscaffold assembly. The alternating gray and white bars represent contiguous scaffolds. Each linkage group is connected to its subsequent chromosome with green horizontal lines which denote SNP positions in the linkage group and its subsequent placement in the superscaffold assembly. Crossing of green horizontal lines indicate points of conflict where the linkage position and superscaffold position are not linear. The ratio between the longest monotonic subsequence and the total number of markers in the linkage group (ρ) indicate high collinearity between SNP marker position and scaffold placement.

Figure 8

Cumulative assembly length. The cumulative lengths of the ALLPATHS-LG contig assembly (black), ALLPATHS-LG scaffold assembly (blue), and the ALLMAPS superscaffold assembly (red) show improvements in total assembly contiguity through this assembly improvement process.

Figure 9

Synteny between B. cucurbitae and D. melanogaster. (A) An analysis for synteny shows how B. cucurbitae (BC) chromosomes correspond to the chromosome arms of D. melanogaster (DM) and the strong conservation of orthologous genes to Muller elements. Orange lines represent orthologous genes between their position on DM chromosome X and their position on BC chromosome 3, blue lines are between DM 2L and BC 5, yellow lines are between DM 2R and BC 6, green lines are between DM 3L and BC 2, purple lines are between DM 3R and BC 4, and red lines are connections between orthologous genes that are not on homologous chromosomes. (B) A dot plot, ordered by Muller element, showing the relationship between gene position in D. melanogaster and the position of its ortholog in B. cucurbitae. A linear regression analysis shows that there is no relationship between the position of an ortholog in a specific D. melanogaster Muller element and the corresponding position of this ortholog in the orthologous B. cucurbitae Muller element, which serves as evidence for widespread intrachromosomal translocations.