Mass releases of sterilized male insects, in the frame of sterile insect technique programs, have helped suppress insect pest populations since the 1950s. In the major horticultural pests Bactrocera dorsalis, Ceratitis capitata, and Zeugodacus cucurbitae, a key phenotype white pupae (wp) has been used for decades to selectively remove females before releases, yet the gene responsible remained unknown. Here, we use classical and modern genetic approaches to identify and functionally characterize causal wp− mutations in these distantly related fruit fly species. We find that the wp phenotype is produced by parallel mutations in a single, conserved gene. CRISPR/Cas9-mediated knockout of the wp gene leads to the rapid generation of white pupae strains in C. capitata and B. tryoni. The conserved phenotype and independent nature of wp− mutations suggest this technique can provide a generic approach to produce sexing strains in other major medical and agricultural insect pests.

AbstractMass releases of sterilized male insects, in the frame of sterile insect technique programs, have helped suppress insect pest populations since the 1950s. In the major horticultural pests Bactrocera dorsalis, Ceratitis capitata, and Zeugodacus cucurbitae, a key phenotype white pupae (wp) has been used for decades to selectively remove females before releases, yet the gene responsible remained unknown. Here we use classical and modern genetic approaches to identify and functionally characterize causal wp− mutations in these distantly related fruit fly species. We find that the wp phenotype is produced by parallel mutations in a single, conserved gene. CRISPR/Cas9-mediated knockout of the wp gene leads to the rapid generation of novel white pupae strains in C. capitata and B. tryoni. The conserved phenotype and independent nature of the wp− mutations suggest that this technique can provide a generic approach to produce sexing strains in other major medical and agricultural insect pests.

We present a genome assembly based on an individual female Aphantopus hyperantus, also known as Maniola hyperantus (the ringlet butterfly; Arthropoda; Insecta; Lepidoptera, Nymphalidae), scaffolded using data from a second, unrelated specimen. The genome sequence is 411 megabases in span. The majority of the assembly is scaffolded into 29 chromosomal pseudomolecules, including the Z sex chromosome.

BackgroundRecent advances in genomics have addressed the challenge that divergent haplotypes pose to the reconstruction of haploid genomes. However for many organisms, the sequencing of either field-caught individuals or a pool of heterogeneous individuals is still the only practical option. Here we present methodological approaches to achieve three outcomes from pooled long read sequencing: the generation of a contiguous haploid reference sequence, the sequences of heterozygous haplotypes; and reconstructed genomic sequences of individuals related to the pooled material. Results PacBio long read sequencing, DovetailHi-C scaffolding and linkage map integration yielded a haploid chromosome-level assembly for the diamondback moth (Plutella xylostella), a global pest of Brassica crops, from a pool of related individuals. The final assembly consisted of 573 scaffolds, with a total assembly size of 343.6Mbp a scaffold N50 value of 11.3Mbp (limited by chromosome size) and a maximum scaffold size of 14.4Mbp. This assembly was then integrated with an existing RAD-seq linkage map, anchoring 95% of the assembled sequence to defined chromosomal positions. ConclusionsWe describe an approach to resolve divergent haplotype sequences and describe multiple validation approaches. We also reconstruct individual genomes from pooled long-reads, by applying a recently developed k-mer binning method.