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Understanding the genetics of adaptation and speciation is critical for a complete picture of how biodiversity is generated and maintained. Heterogeneous genomic differentiation between diverging taxa is commonly documented, with genomic regions of high differentiation interpreted as resulting from differential gene flow, linked selection and reduced recombination rates. Disentangling the roles of each of these non-exclusive processes in shaping genome-wide patterns of divergence is challenging but will enhance our knowledge of the repeatability of genomic landscapes across taxa. Here, we combine whole-genome resequencing and genome feature data to investigate the processes shaping the genomic landscape of differentiation for a sister-species pair of haplodiploid pine sawflies, Neodiprion lecontei and Neodiprion pinetum. We find genome-wide correlations between genome features and summary statistics are consistent with pervasive linked selection, with patterns of diversity and divergence more consistently predicted by exon density and recombination rate than the neutral mutation rate (approximated by dS). We also find that both global and local patterns of FST, dXY and π provide strong support for recurrent selection as the primary selective process shaping variation across pine sawfly genomes, with some contribution from balancing selection and lineage-specific linked selection. Because inheritance patterns for haplodiploid genomes are analogous to those of sex chromosomes, we hypothesize that haplodiploids may be especially prone to recurrent selection, even if gene flow occurred throughout divergence. Overall, our study helps fill an important taxonomic gap in the genomic landscape literature and contributes to our understanding of the processes that shape genome-wide patterns of genetic variation.
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Rapidly evolving taxa are excellent models for understanding the mechanisms that give rise to biodiversity. However, developing an accurate historical framework for comparative analysis of such lineages remains a challenge due to ubiquitous incomplete lineage sorting and introgression. Here, we use a whole-genome alignment, multiple locus-sampling strategies, and summary-tree and SNP-based species-tree methods to infer a species tree for eastern North American Neodiprion species, a clade of pine-feeding sawflies (Order: Hymenopteran; Family: Diprionidae). We recovered a well-supported species tree that-except for three uncertain relationships-was robust to different strategies for analyzing whole-genome data. Nevertheless, underlying gene-tree discordance was high. To understand this genealogical variation, we used multiple linear regression to model site concordance factors estimated in 50-kb windows as a function of several genomic predictor variables. We found that site concordance factors tended to be higher in regions of the genome with more parsimony-informative sites, fewer singletons, less missing data, lower GC content, more genes, lower recombination rates, and lower D-statistics (less introgression). Together, these results suggest that incomplete lineage sorting, introgression, and genotyping error all shape the genomic landscape of gene-tree discordance in Neodiprion. More generally, our findings demonstrate how combining phylogenomic analysis with knowledge of local genomic features can reveal mechanisms that produce topological heterogeneity across genomes.
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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 B. huntii colonies for growers to deliver pollination services to crops. To better understand B. huntii biology and support population genetic studies and breeding decisions, we sequenced and assembled the B. 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 B. 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, showcase efficient techniques for producing a comprehensive, highly contiguous genome.
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The implementation of a new genomic assembly pipeline named only the best (otb) has effectively addressed various challenges associated with data management during the development and storage of genome assemblies. otb, which incorporates a comprehensive pipeline involving a setup layer, quality checks, templating, and the integration of Nextflow and Singularity. The primary objective of otb is to streamline the process of creating a HiFi/HiC genome, aiming to minimize the manual intervention required in the genome assembly process. The 2-lined spittlebug, (Prosapia bicincta, Hemiptera: Cercopidae), a true bug insect herbivore, serves as a practical test case for evaluating otb. The 2-lined spittlebug is both a crucial agricultural pest and a genomically understudied insect belonging to the order Hemiptera. This insect is a significant threat to grasslands and pastures, leading to plant wilting and phytotoxemia when infested. Its presence in tropical and subtropical regions around the world poses a long-term threat to the composition of plant communities in grassland landscapes, impacting rangelands, and posing a substantial risk to cattle production.
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Genoma de Inseto , Genômica , Animais , Genômica/métodos , Hemípteros/genética , SoftwareRESUMO
The West Indian fruit fly, Anastrepha obliqua, is a major pest of mango in Central and South America and attacks more than 60 species of host fruits. To support current genetic and genomic research on A. obliqua, we sequenced the genome using high-fidelity long-read sequencing. This resulted in a highly contiguous contig assembly with 90% of the genome in 10 contigs. The contig assembly was placed in a chromosomal context using synteny with a closely related species, Anastrepha ludens, as both are members of the Anastrepha fraterculus group. The resulting assembly represents the five autosomes and the X chromosome which represents 95.9% of the genome, and 199 unplaced contigs representing the remaining 4.1%. Orthology analysis across the structural annotation sets of high quality tephritid genomes demonstrates the gene annotations are robust, and identified genes unique to Anastrepha species that may help define their pestiferous nature that can be used as a starting point for comparative genomics. This genome assembly represents the first of this species and will serve as a foundation for future genetic and genomic research in support of its management as an agricultural pest.
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Tephritidae , Animais , Tephritidae/genética , Especificidade da Espécie , Drosophila , Frutas , Cromossomo XRESUMO
The Mojave poppy bee, Perdita meconis Griswold (Hymenoptera: Anthophila: Andrenidae), is a species of conservation concern that is restricted to the eastern Mojave Desert of North America. It is a specialist pollinator of two poppy genera, Arctomecon and Argemone (Papaveraceae), and is being considered for listing under the US Endangered Species Act along with one of its pollinator hosts, the Las Vegas bearpoppy (Arctomecon californica). Here, we present a near chromosome-level genome of the Mojave poppy bee to provide a genomic resource that will aid conservation efforts and future research. We isolated DNA from a single, small (<7 mm), male specimen collected using non-ideal preservation methods then performed whole-genome sequencing using PacBio HiFi technology. After quality and contaminant filtering, the final draft genome assembly is 327 Mb, with an N50 length of 17.5 Mb. Annotated repetitive elements compose 37.3% of the genome, although a large proportion (24.87%) of those are unclassified repeats. Additionally, we annotated 18,245 protein-coding genes and 19,433 transcripts. This genome represents one of only a few genomes from the large bee family Andrenidae and one of only a few genomes for pollinator specialists. We highlight both the potential of this genome as a resource for future research, and how high-quality genomes generated from small, non-ideal (in terms of preservation) specimens could facilitate biodiversity genomics.
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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.
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Himenópteros , Humanos , Abelhas/genética , Masculino , Animais , Ecossistema , Espécies em Perigo de Extinção , América do Norte , CromossomosRESUMO
The boll weevil, Anthonomus grandis grandis Boheman, is one of the most historically impactful insects due to its near destruction of the US cotton industry in the early 20th century. Contemporary efforts to manage this insect primarily use pheromone baited traps for detection and organophosphate insecticides for control, but this strategy is not sustainable due to financial and environmental costs. We present a high-quality boll weevil genome assembly, consisting of 306 scaffolds with approximately 24,000 annotated genes, as a first step in the identification of gene targets for novel pest control. Gene content and transposable element distribution are similar to those found in other Curculionidae genomes; however, this is the most contiguous and only assembly reported to date for a member in the species-rich genus Anthonomus. Transcriptome profiles across larval, pupal, and adult life stages led to identification of several genes and gene families that could present targets for novel control strategies.
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Besouros , Inseticidas , Gorgulhos , Animais , Gorgulhos/genética , Besouros/genética , Larva , Biologia , GossypiumRESUMO
Helicoverpa zea (Lepidoptera: Noctuidae) is an insect pest of major cultivated crops in North and South America. The species has adapted to different host plants and developed resistance to several insecticidal agents, including Bacillus thuringiensis (Bt) insecticidal proteins in transgenic cotton and maize. Helicoverpa zea populations persist year-round in tropical and subtropical regions, but seasonal migrations into temperate zones increase the geographic range of associated crop damage. To better understand the genetic basis of these physiological and ecological characteristics, we generated a high-quality chromosome-level assembly for a single H. zea male from Bt-resistant strain, HzStark_Cry1AcR. Hi-C data were used to scaffold an initial 375.2â Mb contig assembly into 30 autosomes and the Z sex chromosome (scaffold N50 = 12.8â Mb and L50 = 14). The scaffolded assembly was error-corrected with a novel pipeline, polishCLR. The mitochondrial genome was assembled through an improved pipeline and annotated. Assessment of this genome assembly indicated 98.8% of the Lepidopteran Benchmark Universal Single-Copy Ortholog set were complete (98.5% as complete single copy). Repetitive elements comprised approximately 29.5% of the assembly with the plurality (11.2%) classified as retroelements. This chromosome-scale reference assembly for H. zea, ilHelZeax1.1, will facilitate future research to evaluate and enhance sustainable crop production practices.
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Bacillus thuringiensis , Inseticidas , Lepidópteros , Mariposas , Animais , Inseticidas/farmacologia , Bacillus thuringiensis/genética , Zea mays , Cromossomos Sexuais , Proteínas de Bactérias/genética , Plantas Geneticamente Modificadas , Proteínas Hemolisinas/genética , Mariposas/genética , Controle Biológico de Vetores , LarvaRESUMO
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.
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Cucurbitaceae , Tephritidae , Animais , Técnicas de Inativação de Genes , Sistemas CRISPR-Cas , Reprodutibilidade dos Testes , Tephritidae/genética , Drosophila/genética , Fenótipo , RecreaçãoRESUMO
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.
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Tephritidae , Animais , Bioensaio , Eugenol/análogos & derivados , Eugenol/farmacologia , Masculino , Comportamento Sexual Animal , Tephritidae/fisiologiaRESUMO
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.
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Sequenciamento de Nucleotídeos em Larga Escala , Software , Biblioteca Gênica , Análise de Sequência de DNARESUMO
BACKGROUND: The small hive beetle (SHB), Aethina tumida, has emerged as a worldwide threat to honey bees in the past two decades. These beetles harvest nest resources, feed on larval bees, and ultimately spoil nest resources with gelatinous slime together with the fungal symbiont Kodamaea ohmeri. RESULTS: Here, we present the first chromosome-level genome assembly for the SHB. With a 99.1% representation of conserved (BUSCO) arthropod genes, this resource enables the study of chemosensory, digestive, and detoxification traits critical for SHB success and possible control. We use this annotated assembly to characterize features of SHB sex chromosomes and a female-skewed primary sex ratio. We also found chromosome fusion and a lower recombination rate in sex chromosomes than in autosomes. CONCLUSIONS: Genome-enabled insights will clarify the traits that allowed this beetle to exploit hive resources successfully and will be critical for determining the causes of observed sex ratio asymmetries.
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Besouros , Parasitos , Animais , Feminino , Abelhas , Larva , Cromossomos Sexuais , Razão de Masculinidade , MasculinoRESUMO
The phylum Arthropoda includes species crucial for ecosystem stability, soil health, crop production, and others that present obstacles to crop and animal agriculture. The United States Department of Agriculture's Agricultural Research Service initiated the Ag100Pest Initiative to generate reference genome assemblies of arthropods that are (or may become) pests to agricultural production and global food security. We describe the project goals, process, status, and future. The first three years of the project were focused on species selection, specimen collection, and the construction of lab and bioinformatics pipelines for the efficient production of assemblies at scale. Contig-level assemblies of 47 species are presented, all of which were generated from single specimens. Lessons learned and optimizations leading to the current pipeline are discussed. The project name implies a target of 100 species, but the efficiencies gained during the project have supported an expansion of the original goal and a total of 158 species are currently in the pipeline. We anticipate that the processes described in the paper will help other arthropod research groups or other consortia considering genome assembly at scale.
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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.
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Proteínas de Insetos/genética , Mutação , Controle Biológico de Vetores/métodos , Pupa/genética , Tephritidae/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Sistemas CRISPR-Cas , Ceratitis capitata/genética , Feminino , Fertilidade/genética , Genoma de Inseto/genética , Masculino , Fenótipo , Reprodução/genética , Tephritidae/classificaçãoRESUMO
Ecological speciation via host-shifting is often invoked as a mechanism for insect diversification, but the relative importance of this process is poorly understood. The shift of Rhagoletis pomonella in the 1850s from the native downy hawthorn, Crataegus mollis, to introduced apple, Malus pumila, is a classic example of sympatric host race formation, a hypothesized early stage of ecological speciation. The accidental human-mediated introduction of R. pomonella into the Pacific Northwest (PNW) in the late 1970s allows us to investigate how novel ecological opportunities may trigger divergent adaptation and host race formation on a rapid timescale. Since the introduction, the fly has spread in the PNW, where in addition to apple, it now infests native black hawthorn, Crataegus douglasii, and introduced ornamental hawthorn, Crataegus monogyna. We use this "natural experiment" to test for genetic differentiation among apple, black, and ornamental hawthorn flies co-occurring at three sympatric sites. We report evidence that populations of all three host-associations are genetically differentiated at the local level, indicating that partial reproductive isolation has evolved in this novel habitat. Our results suggest that conditions suitable for initiating host-associated divergence may be common in nature, allowing for the rapid evolution of new host races when ecological opportunity arises.
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Crataegus , Herbivoria , Malus , Isolamento Reprodutivo , Simpatria , Tephritidae/fisiologia , Animais , Espécies Introduzidas , WashingtonRESUMO
The rice coral, Montipora capitata, is widely distributed throughout the Indo-Pacific and comprises one of the most important reef-building species in the Hawaiian Islands. Here, we describe a de novo assembly of its genome based on a linked-read sequencing approach developed by 10x Genomics. The final draft assembly consisted of 27,870 scaffolds with a N50 size of 186 kb and contained a fairly complete set (81%) of metazoan benchmarking (BUSCO) genes. Based on haploid assembly size (615 Mb) and read k-mer profiles, we estimated the genome size to fall between 600 and 700 Mb, although the high fraction of repetitive sequence introduced considerable uncertainty. Repeat analysis indicated that 42% of the assembly consisted of interspersed, mostly unclassified repeats, and almost 3% tandem repeats. We also identified 36,691 protein-coding genes with a median coding sequence length of 807 bp, together spanning 7% of the assembly. The high repeat content and heterozygosity of the genome proved a challenging scenario for assembly, requiring additional steps to merge haplotypes and resulting in a higher than expected fragmentation at the scaffold level. Despite these challenges, the assembly turned out to be comparable in most quality measures to that of other available coral genomes while being considerably more cost-effective, especially with respect to long-read sequencing methods. Provided high-molecular-weight DNA is available, linked-read technology may thus serve as a valuable alternative capable of providing quality genome assemblies of nonmodel organisms.
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Antozoários/genética , Genoma/genética , Animais , Genômica/métodos , Análise de Sequência de DNARESUMO
Biological control agents have several advantages over chemical control for pest management, including the capability to restore ecosystem balance with minimal non-target effects and a lower propensity for targets to develop resistance. These factors are particularly important for invasive species control. The coconut rhinoceros beetle (Oryctes rhinoceros Linnaeus) is a major palm pest that invaded many Pacific islands in the early 20th century through human-mediated dispersal. Application of the Oryctes nudivirus in the 1960s successfully halted the beetle's first invasion wave and made it a textbook example of successful biological control. However, a recently discovered O. rhinoceros biotype that is resistant to the nudivirus appears to be correlated with a new invasion wave. We performed a population genomics analysis of 172 O. rhinoceros from seven regions, including native and invasive populations, to reconstruct invasion pathways and explore correlation between recent invasions and biotypes. With ddRAD sequencing, we generated data sets ranging from 4,000 to 209,000 loci using stacks and ipyrad software pipelines and compared genetic signal in downstream clustering and phylogenetic analyses. Analysis suggests that the O. rhinoceros resurgence is mediated by the nudivirus-resistant biotype. Genomic data have been proven essential to understanding the new O. rhinoceros biotype's invasion patterns and interactions with the original biotype. Such information is crucial to optimization of strategies for quarantine and control of resurgent pests. Our results demonstrate that while invasions are relatively rare events, new introductions can have significant ecological consequences, and quarantine vigilance is required even in previously invaded areas.
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Cocos , Besouros/genética , Besouros/virologia , Genética Populacional , Controle Biológico de Vetores , Animais , Agentes de Controle Biológico , Resistência à Doença/genética , Espécies Introduzidas , Ilhas do Pacífico , Filogenia , Polimorfismo de Nucleotídeo Único , Análise de Sequência de DNARESUMO
Background: One of the most overlooked, yet critical, components of a whole genome sequencing (WGS) project is the submission and curation of the data to a genomic repository, most commonly the National Center for Biotechnology Information (NCBI). While large genome centers or genome groups have developed software tools for post-annotation assembly filtering, annotation, and conversion into the NCBI's annotation table format, these tools typically require back-end setup and connection to an Structured Query Language (SQL) database and/or some knowledge of programming (Perl, Python) to implement. With WGS becoming commonplace, genome sequencing projects are moving away from the genome centers and into the ecology or biology lab, where fewer resources are present to support the process of genome assembly curation. To fill this gap, we developed software to assess, filter, and transfer annotation and convert a draft genome assembly and annotation set into the NCBI annotation table (.tbl) format, facilitating submission to the NCBI Genome Assembly database. This software has no dependencies, is compatible across platforms, and utilizes a simple command to perform a variety of simple and complex post-analysis, pre-NCBI submission WGS project tasks. Findings: The Genome Annotation Generator is a consistent and user-friendly bioinformatics tool that can be used to generate a .tbl file that is consistent with the NCBI submission pipeline. Conclusions: The Genome Annotation Generator achieves the goal of providing a publicly available tool that will facilitate the submission of annotated genome assemblies to the NCBI. It is useful for any individual researcher or research group that wishes to submit a genome assembly of their study system to the NCBI.