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Background Pacific Biosciences HiFi read technology is currently the industry standard for high accuracy long-read sequencing that has been widely adopted by large sequencing and assembly initiatives for generation of de novo assemblies in non-model organisms. Though adapter contamination filtering is routine in traditional short-read analysis pipelines, it has not been widely adopted for HiFi workflows. Results Analysis of 55 publicly available HiFi datasets revealed that a read-sanitation step to remove sequence artifacts derived from PacBio library preparation from read pools is necessary as adapter sequences can be erroneously integrated into assemblies. Conclusions Here we describe the nature of adapter contaminated reads, their consequences in assembly, and present HiFiAdapterFilt, a simple and memory efficient solution for removing adapter contaminated reads prior to assembly.

Background The Vespa lineage of hornets demonstrate the potential to displace native species and cause significant damage to US apiculture through predation. In spite of introductions in recent years to North America, eradication efforts have prevented the Northern Giant Hornet, Vespa mandarinia , from establishing. Improved genomic resources could offer insight into the traits that define this lineage: large body size variance, adaptability to new environments, and high potential for invasiveness. Results We sequenced and assembled genomes of two lineages of the northern giant hornet, Vespa mandarinia , and one of the European hornet, Vespa crabro , using HiFi long read sequencing technology. We found centromeric and pericentric satellite repeats account for nearly half the total DNA of the hornet genomes and their identities were largely unique across species, indicating active, independent expansion. The intraspecific northern giant hornet genomes exhibit asymmetrical expansion across homologous chromosomal regions localized with Hi-C scaffolding. We leveraged pangenomic alignments of the hornet genomes to identify derived mutations, particularly those that affect repeat content and transposable elements (TEs). We found that TEs do not contribute to the bulk repeat content and show no differential expansion in genic space. Conclusions Large tandemly repetitive DNA account for large structural variations across the Vespa and between V. mandarinia . Localization within the genomes necessitated a suite of tools to support the assembly and identification of those elements. Typical repercussions of long-term reductions in population size – namely, reduced diversity and TE expansion – are not present. The degree to which alternative explanations, such as cell and body size selection or centromeric drive, cause massive, localized repeat amplification will require more extensive sampling.

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. The white pupae (wp) phenotype has been used for decades to selectively remove females of tephritid species in genetic sexing, but the determining gene is unknown. Here, the authors show that wp phenotype is produced by parallel mutations in a Major Facilitator Superfamily domain containing gene across multiple species.

Ongoing climate change has affected the ecological dynamics of many species and is expected to impose natural selection on ecologically important traits. Droughts and other anticipated changes in precipitation may be particularly potent selective factors, especially in arid regions. Here we demonstrate the evolutionary response of an annual plant, Brassica rapa, to a recent climate fluctuation resulting in a multiyear drought. Ancestral (predrought) genotypes were recovered from stored seed and raised under a set of common environments with descendant (postdrought) genotypes and with ancestorxdescendant hybrids. As predicted, the abbreviated growing seasons caused by drought led to the evolution of earlier onset of flowering. Descendants bloomed earlier than ancestors, advancing first flowering by 1.9 days in one study population and 8.6 days in another. The intermediate flowering time of ancestorxdescendant hybrids supports an additive genetic basis for divergence. Experiments confirmed that summer drought selected for early flowering, that flowering time was heritable, and that selection intensities in the field were more than sufficient to account for the observed evolutionary change. Natural selection for drought escape thus appears to have caused adaptive evolution in just a few generations. A systematic effort to collect and store propagules from suitable species would provide biologists with materials to detect and elucidate the genetic basis of further evolutionary shifts driven by climate change.

Hi-C characterizes three-dimensional chromatin organization, facilitates haplotype phasing, and enables genome-assembly scaffolding, but encounters difficulties across complex regions. By coupling chromosome conformation capture (3 C ) with PacBio H iFi long-read sequencing, here we develop a method (CiFi) that enables analysis of genomic interactions across repetitive regions. Starting with as little as 60,000 cells (sub-microgram DNA), the method produces multi-kilobasepair HiFi reads that contain multiple interacting, concatenated segments (~350 bp to 2 kbp). This multiplicity and increase in segment length versus standard short-read-based Hi-C improves read-mapping efficiency and coverage in repetitive regions and enhances haplotype phasing. CiFi pairwise interactions are largely concordant with Hi-C from a human lymphoblastoid cell line, with gains in assigning topologically associating domains across centromeres, segmental duplications, and human disease-associated genomic hotspots. As CiFi requires less input versus established methods, we apply the approach to characterize single small insects: assaying chromatin interactions across the genome from an Anopheles coluzzii mosquito and producing a chromosome-scale scaffolded assembly from a Ceratitis capitata Mediterranean fruit fly. Together, CiFi enables assessment of chromosome-scale interactions of previously recalcitrant low-complexity loci, low-input samples, and small organisms. Hi-C methods for studying 3D genome structure typically require millions of cells and struggle with repetitive regions. Here, authors develop CiFi, combining 3C with PacBio HiFi sequencing, enabling chromatin analysis from as few as 60,000 cells and chromosome-scale assembly from small samples.

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.

Insect responses to chemical attractants are often measured using olfactory bioassays prior to testing in field experiments. The attraction of sexually mature male Bactrocera dorsalis to methyl eugenol (ME) and the loss of attraction by ME pre-fed males have been demonstrated in laboratory bioassays and field trapping studies. It has been suggested that ME nonresponsiveness can be exploited to improve the effectiveness of B. dorsalis management programs by protecting sterile males from ME-based control measures. Currently, work is underway to identify alternatives that reduce or eliminate ME response. To support the development of compounds and evaluation of their effect on B. dorsalis attraction to ME, we compared the effectiveness of three common bioassay methods that have been used to measure lure response in Bactrocera flies under controlled conditions (choice assays using Y-tube [Y], small-cage arena [SC], and rotating carousel field-cage [RC]) to determine which bioassay method is efficient and reliable. A series of bioassays comparing ME-exposed and ME-naïve wild-type and genetic sexing strain males showed that the RC and SC were effective at both observing attraction to ME and detecting a significant reduction in ME response from ME-exposed males. However, the male attraction to ME and a significant decrease in response to ME after ME feeding was not observed in our Y-tube assays. These suggest that RC and SC are preferable options to evaluate ME non-responsiveness in B. dorsalis, and that Y-tube tests are difficult to administer correctly.

The rusty patched bumble bee, Bombus affinis, is an important pollinator in North America and a federally listed endangered species. Due to habitat loss and large declines in population size, B. affinis is facing imminent extinction unless human intervention and recovery efforts are implemented. To better understand B. affinis biology and population genetic and genomic landscapes, we sequenced and assembled the B. affinis genome from a single haploid male. Whole genome HiFi sequencing on PacBio coupled with HiC sequencing resulted in a complete and highly contiguous contig assembly that was scaffolded into a chromosomal context, resolving 18 chromosomes distributed across the 365.1 Mb assembly. All material for both HiFi and HiC sequencing was derived from a single abdominal tissue segment from the single male. These assembly results, coupled with the minimal amount of tissue destructively sampled, demonstrate methods for generating contiguous and complete genomic resources for a rare and endangered species with limited material available and highlight the importance of sample preservation. Precise methods and applications of these methods are presented for potential applications in other species with similar limitations in specimen availability and curation considerations.

Tephritid fruit flies are among the most invasive and destructive agricultural pests worldwide. Over recent years, many studies have implemented the CRISPR/Cas9 genome-editing technology to dissect gene functions in tephritids and create new strains to facilitate their genetics, management, and control. This growing literature allows us to compare diverse strategies for delivering CRISPR/Cas9 components into tephritid embryos, optimize procedures, and advance the technology to systems outside the most thoroughly studied species within the family. Here, we revisit five years of CRISPR research in Tephritidae and propose a unified protocol for candidate gene knockout in fruit flies using CRISPR/Cas9. We demonstrated the efficiency of our protocol by disrupting the eye pigmentation gene white eye (we) in the melon fly, Zeugodacus cucurbitae (Coquillett) (Diptera: Tephritidae). High rates of somatic and germline mutagenesis were induced by microinjecting pre-assembled Cas9-sgRNA complexes through the chorion of embryos at early embryogenesis, leading to the rapid development of new mutant lines. We achieved comparable results when targeting the we orthologue in the oriental fruit fly, Bactrocera dorsalis (Hendel) (Diptera: Tephritidae), illustrating the reliability of our methods when transferred to other related species. Finally, we functionally validated the recently discovered white pupae (wp) loci in the melon fly, successfully recreating the white puparium phenotype used in suppression programs of this and other major economically important tephritids. This is the first demonstration of CRISPR-based genome-editing in the genus Zeugodacus, and we anticipate that the procedures described here will contribute to advancing genome-editing in other non-model tephritid fruit flies.

The Hunt bumble bee, Bombus huntii, is a widely distributed pollinator in western North America. The species produces large colony sizes in captive rearing conditions, experiences low parasite and pathogen loads, and has been demonstrated to be an effective pollinator of tomatoes grown in controlled environment agriculture systems. These desirable traits have galvanized producer efforts to develop commercial Bombus huntii colonies for growers to deliver pollination services to crops. To better understand Bombus huntii biology and support population genetic studies and breeding decisions, we sequenced and assembled the Bombus huntii genome from a single haploid male. High-fidelity sequencing of the entire genome using PacBio, along with HiC sequencing, led to a comprehensive contig assembly of high continuity. This assembly was further organized into a chromosomal arrangement, successfully identifying 18 chromosomes spread across the 317.4 Mb assembly with a BUSCO score indicating 97.6% completeness. Synteny analysis demonstrates shared chromosome number (n = 18) with Bombus terrestris, a species belonging to a different subgenus, matching the expectation that presence of 18 haploid chromosomes is an ancestral trait at least between the subgenera Pyrobombus and Bombus sensu stricto. In conclusion, the assembly outcome, alongside the minimal tissue sampled destructively, showcases efficient techniques for producing a comprehensive, highly contiguous genome.