Galba: genome annotation with miniprot and AUGUSTUS.

BACKGROUND: The Earth Biogenome Project has rapidly increased the number of available eukaryotic genomes, but most released genomes continue to lack annotation of protein-coding genes. In addition, no transcriptome data is available for some genomes. RESULTS: Various gene annotation tools have been developed but each has its limitations. Here, we introduce GALBA, a fully automated pipeline that utilizes miniprot, a rapid protein-to-genome aligner, in combination with AUGUSTUS to predict genes with high accuracy. Accuracy results indicate that GALBA is particularly strong in the annotation of large vertebrate genomes. We also present use cases in insects, vertebrates, and a land plant. GALBA is fully open source and available as a docker image for easy execution with Singularity in high-performance computing environments. CONCLUSIONS: Our pipeline addresses the critical need for accurate gene annotation in newly sequenced genomes, and we believe that GALBA will greatly facilitate genome annotation for diverse organisms.

Chromosome-level genome assemblies of two parasitoid biocontrol wasps reveal the parthenogenesis mechanism and an associated novel virus.

BACKGROUND: Biocontrol is a key technology for the control of pest species. Microctonus parasitoid wasps (Hymenoptera: Braconidae) have been released in Aotearoa New Zealand as biocontrol agents, targeting three different pest weevil species. Despite their value as biocontrol agents, no genome assemblies are currently available for these Microctonus wasps, limiting investigations into key biological differences between the different species and strains. METHODS AND FINDINGS: Here we present high-quality genomes for Microctonus hyperodae and Microctonus aethiopoides, assembled with short read sequencing and Hi-C scaffolding. These assemblies have total lengths of 106.7 Mb for M. hyperodae and 129.2 Mb for M. aethiopoides, with scaffold N50 values of 9 Mb and 23 Mb respectively. With these assemblies we investigated differences in reproductive mechanisms, and association with viruses between Microctonus wasps. Meiosis-specific genes are conserved in asexual Microctonus, with in-situ hybridisation validating expression of one of these genes in the ovaries of asexual Microctonus aethiopoides. This implies asexual reproduction in these Microctonus wasps involves meiosis, with the potential for sexual reproduction maintained. Investigation of viral gene content revealed candidate genes that may be involved in virus-like particle production in M. aethiopoides, as well as a novel virus infecting M. hyperodae, for which a complete genome was assembled. CONCLUSION AND SIGNIFICANCE: These are the first published genomes for Microctonus wasps which have been deployed as biocontrol agents, in Aotearoa New Zealand. These assemblies will be valuable resources for continued investigation and monitoring of these biocontrol systems. Understanding the biology underpinning Microctonus biocontrol is crucial if we are to maintain its efficacy, or in the case of M. hyperodae to understand what may have influenced the significant decline of biocontrol efficacy. The potential for sexual reproduction in asexual Microctonus is significant given that empirical modelling suggests this asexual reproduction is likely to have contributed to biocontrol decline. Furthermore the identification of a novel virus in M. hyperodae highlights a previously unknown aspect of this biocontrol system, which may contribute to premature mortality of the host pest. These findings have potential to be exploited in future in attempt to increase the effectiveness of M. hyperodae biocontrol.

Genomics Reveals Widespread Ecological Speciation in Flightless Insects.

Recent genomic analyses have highlighted parallel divergence in response to ecological gradients, but the extent to which altitude can underpin such repeated speciation remains unclear. Wing reduction and flight loss have apparently evolved repeatedly in montane insect assemblages and have been suggested as important drivers of hexapod diversification. We test this hypothesis using genomic analyses of a widespread wing-polymorphic stonefly species complex in New Zealand. We identified over 50,000 polymorphic genetic markers generated across almost 200 Zelandoperla fenestrata stonefly specimens using a newly generated plecopteran reference genome, to reveal widespread parallel speciation between sympatric full-winged and wing-reduced ecotypes. Rather than the existence of a single, widespread, flightless taxon (Zelandoperla pennulata), evolutionary genomic data reveal that wing-reduced upland lineages have speciated repeatedly and independently from full-winged Z. fenestrata. This repeated evolution of reproductive isolation between local ecotype pairs that lack mitochondrial DNA differentiation suggests that ecological speciation has evolved recently. A cluster of outlier single-nucleotide polymorphisms detected in independently wing-reduced lineages, tightly linked in an approximately 85 kb genomic region that includes the developmental "supergene" doublesex, suggests that this "island of divergence" may play a key role in rapid ecological speciation. [Ecological speciation; genome assembly; genomic island of differentiation; genotyping-by-sequencing; incipient species; plecoptera; wing reduction.].

Genomic signatures of parallel alpine adaptation in recently evolved flightless insects.

Natural selection along elevational gradients has potential to drive predictable adaptations across distinct lineages, but the extent of such repeated evolution remains poorly studied for many widespread alpine taxa. We present parallel genomic analyses of two recently evolved flightless alpine insect lineages to test for molecular signatures of repeated alpine adaptation. Specifically, we compare low-elevation vs. alpine stonefly ecotypes from parallel stream populations in which flightless upland ecotypes have been independently derived. We map 67,922 polymorphic genetic markers, generated across 176 Zelandoperla fenestrata specimens from two independent alpine stream populations in New Zealand's Rock and Pillar Range, to a newly developed plecopteran reference genome. Genome-wide scans revealed 31 regions with outlier single nucleotide polymorphisms (SNPs) differentiating lowland vs. alpine ecotypes in Lug Creek, and 37 regions with outliers differentiating ecotypes in Six Mile Creek. Of these regions, 13% (8/60) yielded outlier SNPs across both within-stream ecotype comparisons, implying comparable genomic shifts contribute to this repeated alpine adaptation. Candidate genes closely linked to repeated outlier regions include several with documented roles in insect wing-development (e.g., dishevelled), suggesting that they may contribute to repeated alpine wing reduction. Additional candidate genes have been shown to influence insect fecundity (e.g., ovo) and lifespan (e.g., Mrp4), implying that they might contribute to life history differentiation between upland and lowland ecotypes. Additional outlier genes have potential roles in the evolution of reproductive isolation among ecotypes (hedgehog and Desaturase 1). These results demonstrate how replicated outlier tests across independent lineages can potentially contribute to the discovery of genes underpinning repeated adaptation.

Expanding the conservation genomics toolbox: Incorporating structural variants to enhance genomic studies for species of conservation concern.

Structural variants (SVs) are large rearrangements (>50 bp) within the genome that impact gene function and the content and structure of chromosomes. As a result, SVs are a significant source of functional genomic variation, that is, variation at genomic regions underpinning phenotype differences, that can have large effects on individual and population fitness. While there are increasing opportunities to investigate functional genomic variation in threatened species via single nucleotide polymorphism (SNP) data sets, SVs remain understudied despite their potential influence on fitness traits of conservation interest. In this future-focused Opinion, we contend that characterizing SVs offers the conservation genomics community an exciting opportunity to complement SNP-based approaches to enhance species recovery. We also leverage the existing literature-predominantly in human health, agriculture and ecoevolutionary biology-to identify approaches for readily characterizing SVs and consider how integrating these into the conservation genomics toolbox may transform the way we manage some of the world's most threatened species.

Select and resequence reveals relative fitness of bacteria in symbiotic and free-living environments.

Assays to accurately estimate relative fitness of bacteria growing in multistrain communities can advance our understanding of how selection shapes diversity within a lineage. Here, we present a variant of the "evolve and resequence" approach both to estimate relative fitness and to identify genetic variants responsible for fitness variation of symbiotic bacteria in free-living and host environments. We demonstrate the utility of this approach by characterizing selection by two plant hosts and in two free-living environments (sterilized soil and liquid media) acting on synthetic communities of the facultatively symbiotic bacterium Ensifer meliloti We find (i) selection that hosts exert on rhizobial communities depends on competition among strains, (ii) selection is stronger inside hosts than in either free-living environment, and (iii) a positive host-dependent relationship between relative strain fitness in multistrain communities and host benefits provided by strains in single-strain experiments. The greatest changes in allele frequencies in response to plant hosts are in genes associated with motility, regulation of nitrogen fixation, and host/rhizobia signaling. The approach we present provides a powerful complement to experimental evolution and forward genetic screens for characterizing selection in bacterial populations, identifying gene function, and surveying the functional importance of naturally occurring genomic variation.

Validating Genome-Wide Association Candidates Controlling Quantitative Variation in Nodulation.

Genome-wide association (GWA) studies offer the opportunity to identify genes that contribute to naturally occurring variation in quantitative traits. However, GWA relies exclusively on statistical association, so functional validation is necessary to make strong claims about gene function. We used a combination of gene-disruption platforms (Tnt1 retrotransposons, hairpin RNA-interference constructs, and CRISPR/Cas9 nucleases) together with randomized, well-replicated experiments to evaluate the function of genes that an earlier GWA study in Medicago truncatula had identified as candidates contributing to variation in the symbiosis between legumes and rhizobia. We evaluated ten candidate genes found in six clusters of strongly associated single nucleotide polymorphisms, selected on the basis of their strength of statistical association, proximity to annotated gene models, and root or nodule expression. We found statistically significant effects on nodule production for three candidate genes, each validated in two independent mutants. Annotated functions of these three genes suggest their contributions to quantitative variation in nodule production occur through processes not previously connected to nodulation, including phosphorous supply and salicylic acid-related defense response. These results demonstrate the utility of GWA combined with reverse mutagenesis technologies to discover and validate genes contributing to naturally occurring variation in quantitative traits. The results highlight the potential for GWA to complement forward genetics in identifying the genetic basis of ecologically and economically important traits.

ODG: Omics database generator - a tool for generating, querying, and analyzing multi-omics comparative databases to facilitate biological understanding.

BACKGROUND: Rapid generation of omics data in recent years have resulted in vast amounts of disconnected datasets without systemic integration and knowledge building, while individual groups have made customized, annotated datasets available on the web with few ways to link them to in-lab datasets. With so many research groups generating their own data, the ability to relate it to the larger genomic and comparative genomic context is becoming increasingly crucial to make full use of the data. RESULTS: The Omics Database Generator (ODG) allows users to create customized databases that utilize published genomics data integrated with experimental data which can be queried using a flexible graph database. When provided with omics and experimental data, ODG will create a comparative, multi-dimensional graph database. ODG can import definitions and annotations from other sources such as InterProScan, the Gene Ontology, ENZYME, UniPathway, and others. This annotation data can be especially useful for studying new or understudied species for which transcripts have only been predicted, and rapidly give additional layers of annotation to predicted genes. In better studied species, ODG can perform syntenic annotation translations or rapidly identify characteristics of a set of genes or nucleotide locations, such as hits from an association study. ODG provides a web-based user-interface for configuring the data import and for querying the database. Queries can also be run from the command-line and the database can be queried directly through programming language hooks available for most languages. ODG supports most common genomic formats as well as generic, easy to use tab-separated value format for user-provided annotations. CONCLUSIONS: ODG is a user-friendly database generation and query tool that adapts to the supplied data to produce a comparative genomic database or multi-layered annotation database. ODG provides rapid comparative genomic annotation and is therefore particularly useful for non-model or understudied species. For species for which more data are available, ODG can be used to conduct complex multi-omics, pattern-matching queries.

Exploring structural variation and gene family architecture with De Novo assemblies of 15 Medicago genomes.

BACKGROUND: Previous studies exploring sequence variation in the model legume, Medicago truncatula, relied on mapping short reads to a single reference. However, read-mapping approaches are inadequate to examine large, diverse gene families or to probe variation in repeat-rich or highly divergent genome regions. De novo sequencing and assembly of M. truncatula genomes enables near-comprehensive discovery of structural variants (SVs), analysis of rapidly evolving gene families, and ultimately, construction of a pan-genome. RESULTS: Genome-wide synteny based on 15 de novo M. truncatula assemblies effectively detected different types of SVs indicating that as much as 22% of the genome is involved in large structural changes, altogether affecting 28% of gene models. A total of 63 million base pairs (Mbp) of novel sequence was discovered, expanding the reference genome space for Medicago by 16%. Pan-genome analysis revealed that 42% (180 Mbp) of genomic sequences is missing in one or more accession, while examination of de novo annotated genes identified 67% (50,700) of all ortholog groups as dispensable - estimates comparable to recent studies in rice, maize and soybean. Rapidly evolving gene families typically associated with biotic interactions and stress response were found to be enriched in the accession-specific gene pool. The nucleotide-binding site leucine-rich repeat (NBS-LRR) family, in particular, harbors the highest level of nucleotide diversity, large effect single nucleotide change, protein diversity, and presence/absence variation. However, the leucine-rich repeat (LRR) and heat shock gene families are disproportionately affected by large effect single nucleotide changes and even higher levels of copy number variation. CONCLUSIONS: Analysis of multiple M. truncatula genomes illustrates the value of de novo assemblies to discover and describe structural variation, something that is often under-estimated when using read-mapping approaches. Comparisons among the de novo assemblies also indicate that different large gene families differ in the architecture of their structural variation.

Transcriptomic basis of genome by genome variation in a legume-rhizobia mutualism.

In the legume-rhizobia mutualism, the benefit each partner derives from the other depends on the genetic identity of both host and rhizobial symbiont. To gain insight into the extent of genome × genome interactions on hosts at the molecular level and to identify potential mechanisms responsible for the variation, we examined host gene expression within nodules (the plant organ where the symbiosis occurs) of four genotypes of Medicago truncatula grown with either Ensifer meliloti or E. medicae symbionts. These host × symbiont combinations show significant variation in nodule and biomass phenotypes. Likewise, combinations differ in their transcriptomes: host, symbiont and host × symbiont affected the expression of 70%, 27% and 21%, respectively, of the approximately 27,000 host genes expressed in nodules. Genes with the highest levels of expression often varied between hosts and/or symbiont strain and include leghemoglobins that modulate oxygen availability and hundreds of Nodule Cysteine-Rich (NCR) peptides involved in symbiont differentiation and viability in nodules. Genes with host × symbiont-dependent expression were enriched for functions related to resource exchange between partners (sulphate/iron/amino acid transport and dicarboxylate/amino acid synthesis). These enrichments suggest mechanisms for host control of the currencies of the mutualism. The transcriptome of M. truncatula accession HM101 (A17), the reference genome used for most molecular research, was less affected by symbiont identity than the other hosts. These findings underscore the importance of assessing the molecular basis of variation in ecologically important traits, particularly those involved in biotic interactions, in multiple genetic contexts.

Detecting small plant peptides using SPADA (Small Peptide Alignment Discovery Application).

BACKGROUND: Small peptides encoded as one- or two-exon genes in plants have recently been shown to affect multiple aspects of plant development, reproduction and defense responses. However, popular similarity search tools and gene prediction techniques generally fail to identify most members belonging to this class of genes. This is largely due to the high sequence divergence among family members and the limited availability of experimentally verified small peptides to use as training sets for homology search and ab initio prediction. Consequently, there is an urgent need for both experimental and computational studies in order to further advance the accurate prediction of small peptides. RESULTS: We present here a homology-based gene prediction program to accurately predict small peptides at the genome level. Given a high-quality profile alignment, SPADA identifies and annotates nearly all family members in tested genomes with better performance than all general-purpose gene prediction programs surveyed. We find numerous mis-annotations in the current Arabidopsis thaliana and Medicago truncatula genome databases using SPADA, most of which have RNA-Seq expression support. We also show that SPADA works well on other classes of small secreted peptides in plants (e.g., self-incompatibility protein homologues) as well as non-secreted peptides outside the plant kingdom (e.g., the alpha-amanitin toxin gene family in the mushroom, Amanita bisporigera). CONCLUSIONS: SPADA is a free software tool that accurately identifies and predicts the gene structure for short peptides with one or two exons. SPADA is able to incorporate information from profile alignments into the model prediction process and makes use of it to score different candidate models. SPADA achieves high sensitivity and specificity in predicting small plant peptides such as the cysteine-rich peptide families. A systematic application of SPADA to other classes of small peptides by research communities will greatly improve the genome annotation of different protein families in public genome databases.

Germline progenitors and oocyte production in the honeybee queen ovary.

Understanding the reproduction of honeybee queens is crucial to support populations of this economically important insect. Here we examine the structure of the honeybee ovary to determine the nature of the germline progenitors in the ovary. Using a panel of marker genes that mark somatic or germline tissue in other insects we determine which cells in the honeybee ovary are somatic and which germline. We examine patterns of cell division and demonstrate that, unlike Drosophila, there is no evidence of single germline stem cells that provide the germline in honeybees. Germline progenitors are clustered in groups of 8 cells, joined by a polyfusome, and collections of these, in each ovariole, appear to maintain the germline during reproduction. We also show that these 8-cell clusters can divide and that their division occurs such that the numbers of germline progenitors are relatively constant over the reproductive life of queen honeybees. This information helps us to understand the diversity of structures in insect reproduction, and provide information to better support honeybee reproduction.

The promise and challenges of characterizing genome-wide structural variants: A case study in a critically endangered parrot.

There is growing interest in the role of structural variants (SVs) as drivers of local adaptation and speciation. From a biodiversity genomics perspective, the characterization of genome-wide SVs provides an exciting opportunity to complement single nucleotide polymorphisms (SNPs). However, little is known about the impacts of SV discovery and genotyping strategies on the characterization of genome-wide SV diversity within and among populations. Here, we explore a near whole-species resequence data set, and long-read sequence data for a subset of highly represented individuals in the critically endangered kakapo (Strigops habroptilus). We demonstrate that even when using a highly contiguous reference genome, different discovery and genotyping strategies can significantly impact the type, size and location of SVs characterized genome-wide. Further, we found that the mean number of SVs in each of two kakapo lineages differed both within and across generations. These combined results suggest that genome-wide characterization of SVs remains challenging at the population-scale. We are optimistic that increased accessibility to long-read sequencing and advancements in bioinformatic approaches including multireference approaches like genome graphs will alleviate at least some of the challenges associated with resolving SV characteristics below the species level. In the meantime, we address caveats, highlight considerations, and provide recommendations for the characterization of genome-wide SVs in biodiversity genomic research.

GALBA: Genome Annotation with Miniprot and AUGUSTUS.

The Earth Biogenome Project has rapidly increased the number of available eukaryotic genomes, but most released genomes continue to lack annotation of protein-coding genes. In addition, no transcriptome data is available for some genomes. Various gene annotation tools have been developed but each has its limitations. Here, we introduce GALBA, a fully automated pipeline that utilizes miniprot, a rapid protein- to-genome aligner, in combination with AUGUSTUS to predict genes with high accuracy. Accuracy results indicate that GALBA is particularly strong in the annotation of large vertebrate genomes. We also present use cases in insects, vertebrates, and a previously unannotated land plant. GALBA is fully open source and available as a docker image for easy execution with Singularity in high-performance computing environments. Our pipeline addresses the critical need for accurate gene annotation in newly sequenced genomes, and we believe that GALBA will greatly facilitate genome annotation for diverse organisms.

Assembly of female and male hihi genomes (stitchbird; Notiomystis cincta) enables characterization of the W chromosome and resources for conservation genomics.

A high-quality reference genome can be a valuable resource for threatened species by providing a foundation to assess their evolutionary potential to adapt to future pressures such as environmental change. We assembled the genome of a female hihi (Notiomysits cincta), a threatened passerine bird endemic to Aotearoa New Zealand. The assembled genome is 1.06 Gb, and is of high quality and highly contiguous, with a contig N50 of 7.0 Mb, estimated QV of 44 and a BUSCO completeness of 96.8%. A male assembly of comparable quality was generated in parallel. A population linkage map was used to scaffold the autosomal contigs into chromosomes. Female and male sequence coverage and comparative genomics analyses were used to identify Z-, and W-linked contigs. In total, 94.6% of the assembly length was assigned to putative nuclear chromosome scaffolds. Native DNA methylation was highly correlated between sexes, with the W chromosome contigs more highly methylated than autosomal chromosomes and Z contigs. 43 differentially methylated regions were identified, and these may represent interesting candidates for the establishment or maintenance of sex differences. By generating a high-quality reference assembly of the heterogametic sex, we have created a resource that enables characterization of genome-wide diversity and facilitates the investigation of female-specific evolutionary processes. The reference genomes will form the basis for fine-scale assessment of the impacts of low genetic diversity and inbreeding on the adaptive potential of the species and will therefore enable tailored and informed conservation management of this threatened taonga (treasured) species.

Hidden impacts of conservation management on fertility of the critically endangered kakapo.

Background: Animal conservation often requires intensive management actions to improve reproductive output, yet any adverse effects of these may not be immediately apparent, particularly in threatened species with small populations and long lifespans. Hand-rearing is an example of a conservation management strategy which, while boosting populations, can cause long-term demographic and behavioural problems. It is used in the recovery of the critically endangered kakapo (Strigops habroptilus), a flightless parrot endemic to New Zealand, to improve the slow population growth that is due to infrequent breeding, low fertility and low hatching success. Methods: We applied Bayesian mixed models to examine whether hand-rearing and other factors were associated with clutch fertility in kakapo. We used projection predictive variable selection to compare the relative contributions to fertility from the parents' rearing environment, their age and previous copulation experience, the parental kinship, and the number of mates and copulations for each clutch. We also explored how the incidence of repeated copulations and multiple mates varied with kakapo density. Results: The rearing status of the clutch father and the number of mates and copulations of the clutch mother were the dominant factors in predicting fertility. Clutches were less likely to be fertile if the father was hand-reared compared to wild-reared, but there was no similar effect for mothers. Clutches produced by females copulating with different males were more likely to be fertile than those from repeated copulations with one male, which in turn had a higher probability of fertility than those from a single copulation. The likelihood of multiple copulations and mates increased with female:male adult sex ratio, perhaps as a result of mate guarding by females. Parental kinship, copulation experience and age all had negligible associations with clutch fertility. Conclusions: These results provide a rare assessment of factors affecting fertility in a wild threatened bird species, with implications for conservation management. The increased fertility due to multiple mates and copulations, combined with the evidence for mate guarding and previous results of kakapo sperm morphology, suggests that an evolutionary mechanism exists to optimise fertility through sperm competition in kakapo. The high frequency of clutches produced from single copulations in the contemporary population may therefore represent an unnatural state, perhaps due to too few females. This suggests that opportunity for sperm competition should be maximised by increasing population densities, optimising sex ratios, and using artificial insemination. The lower fertility of hand-reared males may result from behavioural defects due to lack of exposure to conspecifics at critical development stages, as seen in other taxa. This potential negative impact of hand-rearing must be balanced against the short-term benefits it provides.

Species-wide genomics of kakapo provides tools to accelerate recovery.

The kakapo is a critically endangered, intensively managed, long-lived nocturnal parrot endemic to Aotearoa New Zealand. We generated and analysed whole-genome sequence data for nearly all individuals living in early 2018 (169 individuals) to generate a high-quality species-wide genetic variant callset. We leverage extensive long-term metadata to quantify genome-wide diversity of the species over time and present new approaches using probabilistic programming, combined with a phenotype dataset spanning five decades, to disentangle phenotypic variance into environmental and genetic effects while quantifying uncertainty in small populations. We find associations for growth, disease susceptibility, clutch size and egg fertility within genic regions previously shown to influence these traits in other species. Finally, we generate breeding values to predict phenotype and illustrate that active management over the past 45 years has maintained both genome-wide diversity and diversity in breeding values and, hence, evolutionary potential. We provide new pathways for informing future conservation management decisions for kakapo, including prioritizing individuals for translocation and monitoring individuals with poor growth or high disease risk. Overall, by explicitly addressing the challenge of the small sample size, we provide a template for the inclusion of genomic data that will be transformational for species recovery efforts around the globe.

An exploration of assembly strategies and quality metrics on the accuracy of the rewarewa (Knightia excelsa) genome.

We used long read sequencing data generated from Knightia excelsa, a nectar-producing Proteaceae tree endemic to Aotearoa (New Zealand), to explore how sequencing data type, volume and workflows can impact final assembly accuracy and chromosome reconstruction. Establishing a high-quality genome for this species has specific cultural importance to Maori and commercial importance to honey producers in Aotearoa. Assemblies were produced by five long read assemblers using data subsampled based on read lengths, two polishing strategies and two Hi-C mapping methods. Our results from subsampling the data by read length showed that each assembler tested performed differently depending on the coverage and the read length of the data. Subsampling highlighted that input data with longer read lengths but perhaps lower coverage constructed more contiguous, kmers and gene-complete assemblies than short read length input data with higher coverage. The final genome assembly was constructed into 14 pseudochromosomes using an initial flye long read assembly, a racon/medaka/pilon combined polishing strategy, salsa2 and allhic scaffolding, juicebox curation, and Macadamia linkage map validation. We highlighted the importance of developing assembly workflows based on the volume and read length of sequencing data and established a robust set of quality metrics for generating high-quality assemblies. Scaffolding analyses highlighted that problems found in the initial assemblies could not be resolved accurately by Hi-C data and that assembly scaffolding was more successful when the underlying contig assembly was of higher accuracy. These findings provide insight into how quality assessment tools can be implemented throughout genome assembly pipelines to inform the de novo reconstruction of a high-quality genome assembly for nonmodel organisms.

Population genomics of the critically endangered kakapo.

The kakapo is a flightless parrot endemic to New Zealand. Once common in the archipelago, only 201 individuals remain today, most of them descending from an isolated island population. We report the first genome-wide analyses of the species, including a high-quality genome assembly for kakapo, one of the first chromosome-level reference genomes sequenced by the Vertebrate Genomes Project (VGP). We also sequenced and analyzed 35 modern genomes from the sole surviving island population and 14 genomes from the extinct mainland population. While theory suggests that such a small population is likely to have accumulated deleterious mutations through genetic drift, our analyses on the impact of the long-term small population size in kakapo indicate that present-day island kakapo have a reduced number of harmful mutations compared to mainland individuals. We hypothesize that this reduced mutational load is due to the island population having been subjected to a combination of genetic drift and purging of deleterious mutations, through increased inbreeding and purifying selection, since its isolation from the mainland ∼10,000 years ago. Our results provide evidence that small populations can survive even when isolated for hundreds of generations. This work provides key insights into kakapo breeding and recovery and more generally into the application of genetic tools in conservation efforts for endangered species.

The potential for a CRISPR gene drive to eradicate or suppress globally invasive social wasps.

CRISPR gene drives have potential for widespread and cost-efficient pest control, but are highly controversial. We examined a potential gene drive targeting spermatogenesis to control the invasive common wasp (Vespula vulgaris) in New Zealand. Vespula wasps are haplodiploid. Their life cycle makes gene drive production challenging, as nests are initiated by single fertilized queens in spring followed by several cohorts of sterile female workers and the production of reproductives in autumn. We show that different spermatogenesis genes have different levels of variation between introduced and native ranges, enabling a potential 'precision drive' that could target the reduced genetic diversity and genotypes within the invaded range. In vitro testing showed guide-RNA target specificity and efficacy that was dependent on the gene target within Vespula, but no cross-reactivity in other Hymenoptera. Mathematical modelling incorporating the genetic and life history traits of Vespula wasps identified characteristics for a male sterility drive to achieve population control. There was a trade-off between drive infiltration and impact: a drive causing complete male sterility would not spread, while partial sterility could be effective in limiting population size if the homing rate is high. Our results indicate that gene drives may offer viable suppression for wasps and other haplodiploid pests.