The complete genome assemblies of 19 insect pests of worldwide importance to agriculture.

There are many insect pests worldwide that damage agricultural crop and reduce yield either by direct feeding or by the transmission of plant diseases. To date, control of pest insects has been achieved largely by applying synthetic insecticides. However, insecticide use can be seriously impacted by legislation that limits their use or by the evolution of resistance in the target pest. Thus, there is a move towards less use of insecticides and increased adoption of integrated pest management strategies using a wide range of non-chemical and chemical control methods. For good pest control there is a need to understand the mode of action and selectivity of insecticides, the life cycles of the pests and their biology and behaviours, all of which can benefit from good quality genome data. Here we present the complete assembled (chromosome level) genomes (incl. mtDNA) of 19 insect pests, Agriotes lineatus (click beetle/wireworm), Aphis gossypii (melon/cotton aphid), Bemisia tabaci (cotton whitefly), Brassicogethes aeneus (pollen beetle), Ceutorhynchus obstrictus (seedpod weevil), Chilo suppressalis (striped rice stem borer), Chrysodeixis includens (soybean looper), Diabrotica balteata (cucumber beetle), Diatraea saccharalis (sugar cane borer), Nezara viridula (green stink bug), Nilaparvata lugens (brown plant hopper), Phaedon cochleariae (mustard beetle), Phyllotreta striolata (striped flea beetle), Psylliodes chrysocephala (cabbage stem flea beetle), Spodoptera exigua (beet army worm), Spodoptera littoralis (cotton leaf worm), Diabrotica virgifera (western corn root worm), Euschistus heros (brown stink bug) and Phyllotreta cruciferae (crucifer flea beetle). For the first 15 of these we also present the annotation of genes encoding potential xenobiotic detoxification enzymes. This public resource will aid in the elucidation and monitoring of resistance mechanisms, the development of highly selective chemistry and potential techniques to disrupt behaviour in a way that limits the effect of the pests.

A scaffold-level genome assembly of a minute pirate bug, Orius laevigatus (Hemiptera: Anthocoridae), and a comparative analysis of insecticide resistance-related gene families with hemipteran crop pests.

BACKGROUND: Orius laevigatus, a minute pirate bug, is a highly effective beneficial predator of crop pests including aphids, spider mites and thrips in integrated pest management (IPM) programmes. No genomic information is currently available for O. laevigatus, as is the case for the majority of beneficial predators which feed on crop pests. In contrast, genomic information for crop pests is far more readily available. The lack of publicly available genomes for beneficial predators to date has limited our ability to perform comparative analyses of genes encoding potential insecticide resistance mechanisms between crop pests and their predators. These mechanisms include several gene/protein families including cytochrome P450s (P450s), ATP binding cassette transporters (ABCs), glutathione S-transferases (GSTs), UDP-glucosyltransferases (UGTs) and carboxyl/cholinesterases (CCEs). METHODS AND FINDINGS: In this study, a high-quality scaffold level de novo genome assembly for O. laevigatus has been generated using a hybrid approach with PacBio long-read and Illumina short-read data. The final assembly achieved a scaffold N50 of 125,649 bp and a total genome size of 150.98 Mb. The genome assembly achieved a level of completeness of 93.6% using a set of 1658 core insect genes present as full-length genes. Genome annotation identified 15,102 protein-coding genes - 87% of which were assigned a putative function. Comparative analyses revealed gene expansions of sigma class GSTs and CYP3 P450s. Conversely the UGT gene family showed limited expansion. Differences were seen in the distributions of resistance-associated gene families at the subfamily level between O. laevigatus and some of its targeted crop pests. A target site mutation in ryanodine receptors (I4790M, PxRyR) which has strong links to diamide resistance in crop pests and had previously only been identified in lepidopteran species was found to also be present in hemipteran species, including O. laevigatus. CONCLUSION AND SIGNIFICANCE: This assembly is the first published genome for the Anthocoridae family and will serve as a useful resource for further research into target-site selectivity issues and potential resistance mechanisms in beneficial predators. Furthermore, the expansion of gene families often linked to insecticide resistance may be an indicator of the capacity of this predator to detoxify selective insecticides. These findings could be exploited by targeted pesticide screens and functional studies to increase effectiveness of IPM strategies, which aim to increase crop yields by sustainably, environmentally-friendly and effectively control pests without impacting beneficial predator populations.

A near-chromosome level genome assembly of the European hoverfly, Sphaerophoria rueppellii (Diptera: Syrphidae), provides comparative insights into insecticide resistance-related gene family evolution.

BACKGROUND: Sphaerophoria rueppellii, a European species of hoverfly, is a highly effective beneficial predator of hemipteran crop pests including aphids, thrips and coleopteran/lepidopteran larvae in integrated pest management (IPM) programmes. It is also a key pollinator of a wide variety of important agricultural crops. No genomic information is currently available for S. rueppellii. Without genomic information for such beneficial predator species, we are unable to perform comparative analyses of insecticide target-sites and genes encoding metabolic enzymes potentially responsible for insecticide resistance, between crop pests and their predators. These metabolic mechanisms include several gene families - cytochrome P450 monooxygenases (P450s), ATP binding cassette transporters (ABCs), glutathione-S-transferases (GSTs), UDP-glycosyltransferases (UGTs) and carboxyl/choline esterases (CCEs). METHODS AND FINDINGS: In this study, a high-quality near-chromosome level de novo genome assembly (as well as a mitochondrial genome assembly) for S. rueppellii has been generated using a hybrid approach with PacBio long-read and Illumina short-read data, followed by super scaffolding using Hi-C data. The final assembly achieved a scaffold N50 of 87Mb, a total genome size of 537.6Mb and a level of completeness of 96% using a set of 1,658 core insect genes present as full-length genes. The assembly was annotated with 14,249 protein-coding genes. Comparative analysis revealed gene expansions of CYP6Zx P450s, epsilon-class GSTs, dietary CCEs and multiple UGT families (UGT37/302/308/430/431). Conversely, ABCs, delta-class GSTs and non-CYP6Zx P450s showed limited expansion. Differences were seen in the distributions of resistance-associated gene families across subfamilies between S. rueppellii and some hemipteran crop pests. Additionally, S. rueppellii had larger numbers of detoxification genes than other pollinator species. CONCLUSION AND SIGNIFICANCE: This assembly is the first published genome for a predatory member of the Syrphidae family and will serve as a useful resource for further research into selectivity and potential tolerance of insecticides by beneficial predators. Furthermore, the expansion of some gene families often linked to insecticide resistance and selectivity may be an indicator of the capacity of this predator to detoxify IPM selective insecticides. These findings could be exploited by targeted insecticide screens and functional studies to increase effectiveness of IPM strategies, which aim to increase crop yields by sustainably and effectively controlling pests without impacting beneficial predator populations.

Exploring genetic variation in the tomato (Solanum section Lycopersicon) clade by whole-genome sequencing.

We explored genetic variation by sequencing a selection of 84 tomato accessions and related wild species representative of the Lycopersicon, Arcanum, Eriopersicon and Neolycopersicon groups, which has yielded a huge amount of precious data on sequence diversity in the tomato clade. Three new reference genomes were reconstructed to support our comparative genome analyses. Comparative sequence alignment revealed group-, species- and accession-specific polymorphisms, explaining characteristic fruit traits and growth habits in the various cultivars. Using gene models from the annotated Heinz 1706 reference genome, we observed differences in the ratio between non-synonymous and synonymous SNPs (dN/dS) in fruit diversification and plant growth genes compared to a random set of genes, indicating positive selection and differences in selection pressure between crop accessions and wild species. In wild species, the number of single-nucleotide polymorphisms (SNPs) exceeds 10 million, i.e. 20-fold higher than found in most of the crop accessions, indicating dramatic genetic erosion of crop and heirloom tomatoes. In addition, the highest levels of heterozygosity were found for allogamous self-incompatible wild species, while facultative and autogamous self-compatible species display a lower heterozygosity level. Using whole-genome SNP information for maximum-likelihood analysis, we achieved complete tree resolution, whereas maximum-likelihood trees based on SNPs from ten fruit and growth genes show incomplete resolution for the crop accessions, partly due to the effect of heterozygous SNPs. Finally, results suggest that phylogenetic relationships are correlated with habitat, indicating the occurrence of geographical races within these groups, which is of practical importance for Solanum genome evolution studies.

Systematic identification of balanced transposition polymorphisms in Saccharomyces cerevisiae.

High-throughput techniques for detecting DNA polymorphisms generally do not identify changes in which the genomic position of a sequence, but not its copy number, varies among individuals. To explore such balanced structural polymorphisms, we used array-based Comparative Genomic Hybridization (aCGH) to conduct a genome-wide screen for single-copy genomic segments that occupy different genomic positions in the standard laboratory strain of Saccharomyces cerevisiae (S90) and a polymorphic wild isolate (Y101) through analysis of six tetrads from a cross of these two strains. Paired-end high-throughput sequencing of Y101 validated four of the predicted rearrangements. The transposed segments contained one to four annotated genes each, yet crosses between S90 and Y101 yielded mostly viable tetrads. The longest segment comprised 13.5 kb near the telomere of chromosome XV in the S288C reference strain and Southern blotting confirmed its predicted location on chromosome IX in Y101. Interestingly, inter-locus crossover events between copies of this segment occurred at a detectable rate. The presence of low-copy repetitive sequences at the junctions of this segment suggests that it may have arisen through ectopic recombination. Our methodology and findings provide a starting point for exploring the origins, phenotypic consequences, and evolutionary fate of this largely unexplored form of genomic polymorphism.

Evidence for the adaptive significance of an LTR retrotransposon sequence in a Drosophila heterochromatic gene.

BACKGROUND: The potential adaptive significance of transposable elements (TEs) to the host genomes in which they reside is a topic that has been hotly debated by molecular evolutionists for more than two decades. Recent genomic analyses have demonstrated that TE fragments are associated with functional genes in plants and animals. These findings suggest that TEs may contribute significantly to gene evolution. RESULTS: We have analyzed two transposable elements associated with genes in the sequenced Drosophila melanogaster y; cn bw sp strain. A fragment of the Antonia long terminal repeat (LTR) retrotransposon is present in the intron of Chitinase 3 (Cht3), a gene located within the constitutive heterochromatin of chromosome 2L. Within the euchromatin of chromosome 2R a full-length Burdock LTR retrotransposon is located immediately 3' to cathD, a gene encoding cathepsin D. We tested for the presence of these two TE/gene associations in strains representing 12 geographically diverse populations of D. melanogaster. While the cathD insertion variant was detected only in the sequenced y; cn bw sp strain, the insertion variant present in the heterochromatic Cht3 gene was found to be fixed throughout twelve D. melanogaster populations and in a D. mauritiana strain suggesting that it maybe of adaptive significance. To further test this hypothesis, we sequenced a 685bp region spanning the LTR fragment in the intron of Cht3 in strains representative of the two sibling species D. melanogaster and D. mauritiana (approximately 2.7 million years divergent). The level of sequence divergence between the two species within this region was significantly lower than expected from the neutral substitution rate and lower than the divergence observed between a randomly selected intron of the Drosophila Alcohol dehydrogenase gene (Adh). CONCLUSIONS: Our results suggest that a 359 bp fragment of an Antonia retrotransposon (complete LTR is 659 bp) located within the intron of the Drosophila melanogaster Cht3 gene is of adaptive evolutionary significance. Our results are consistent with previous suggestions that the presence of TEs in constitutive heterochromatin may be of significance to the expression of heterochromatic genes.

Evolutionary history of Oryza sativa LTR retrotransposons: a preliminary survey of the rice genome sequences.

BACKGROUND: LTR Retrotransposons transpose through reverse transcription of an RNA intermediate and are ubiquitous components of all eukaryotic genomes thus far examined. Plant genomes, in particular, have been found to be comprised of a remarkably high number of LTR retrotransposons. There is a significant body of direct and indirect evidence that LTR retrotransposons have contributed to gene and genome evolution in plants. RESULTS: To explore the evolutionary history of long terminal repeat (LTR) retrotransposons and their impact on the genome of Oryza sativa, we have extended an earlier computer-based survey to include all identifiable full-length, fragmented and solo LTR elements in the rice genome database as of April 2002. A total of 1,219 retroelement sequences were identified, including 217 full-length elements, 822 fragmented elements, and 180 solo LTRs. In order to gain insight into the chromosomal distribution of LTR-retrotransposons in the rice genome, a detailed examination of LTR-retrotransposon sequences on Chromosome 10 was carried out. An average of 22.3 LTR-retrotransposons per Mb were detected in Chromosome 10. CONCLUSIONS: Gypsy-like elements were found to be >4 x more abundant than copia-like elements. Eleven of the thirty-eight investigated LTR-retrotransposon families displayed significant subfamily structure. We estimate that at least 46.5% of LTR-retrotransposons in the rice genome are older than the age of the species (< 680,000 years). LTR-retrotransposons present in the rice genome range in age from those just recently inserted up to nearly 10 million years old. Approximately 20% of LTR retrotransposon sequences lie within putative genes. The distribution of elements across chromosome 10 is non-random with the highest density (48 elements per Mb) being present in the pericentric region.

Divergence in expression between duplicated genes in Arabidopsis.

New genes may arise through tandem duplication, dispersed small-scale duplication, and polyploidy, and patterns of divergence between duplicated genes may vary among these classes. We have examined patterns of gene expression and coding sequence divergence between duplicated genes in Arabidopsis thaliana. Due to the simultaneous origin of polyploidy-derived gene pairs, we can compare covariation in the rates of expression divergence and sequence divergence within this group. Among tandem and dispersed duplicates, much of the divergence in expression profile appears to occur at or shortly after duplication. Contrary to findings from other eukaryotic systems, there is little relationship between expression divergence and synonymous substitutions, whereas there is a strong positive relationship between expression divergence and nonsynonymous substitutions. Because this pattern is pronounced among the polyploidy-derived pairs, we infer that the strength of purifying selection acting on protein sequence and expression pattern is correlated. The polyploidy-derived pairs are somewhat atypical in that they have broader expression patterns and are expressed at higher levels, suggesting differences among polyploidy- and nonpolyploidy-derived duplicates in the types of genes that revert to single copy. Finally, within many of the duplicated pairs, 1 gene is expressed at a higher level across all assayed conditions, which suggests that the subfunctionalization model for duplicate gene preservation provides, at best, only a partial explanation for the patterns of expression divergence between duplicated genes.

The contribution of LTR retrotransposon sequences to gene evolution in Mus musculus.

Approximately 1.5% of mouse genes (Mus musculus) contain long terminal repeat retrotransposon sequences (LRS). Consistent with earlier findings in Caenorhabditis elegans, Drosophila melanogaster, and Homo sapiens, LRS are more likely to be associated with newly evolved genes. Evidence is presented that LRS are often recruited as novel exons or as spliced additions to existing exons. These novel gene configurations may be expressed initially as alternative transcripts providing an opportunity for the evolution of new gene function.

LTR retrotransposon-gene associations in Drosophila melanogaster.

Thirty-three percent (228/682) of all long terminal repeat (LTR) retrotransposon sequences (LRSs) present in the sequenced Drosophila melanogaster genome were found to be located in or within 1000 bp of a gene. Recently inserted LTR retrotransposons are significantly more likely to be located in or within genes than are older, fragmented LTR retrotransposon sequences, indicating that most LRS-gene associations are selected against over evolutionary time. LRSs associated with conserved genes (homologenes) are especially prone to negative selection. In contrast, fragmented LRSs that have persisted in the genome over long spans of evolutionary time are preferentially associated with genes involved in signal transduction and other newly evolved functions.

Retrotransposon-gene associations are widespread among D. melanogaster populations.

We have surveyed 18 natural populations of Drosophila melanogaster for the presence of 23 retrotransposon-gene-association alleles (i.e., the presence of an LTR retrotransposon sequence in or within 1,000 bp of a gene) recently identified in the sequenced D. melanogaster genome. The identified associations were detected only in the D. melanogaster populations. The majority (61%) of the identified retrotransposon-gene associations were present only in the sequenced strain in which they were first identified. Thirty percent of the associations were detected in at least one of the natural populations, and 9% of the associations were detected in all of the D. melanogaster populations surveyed. Sequence analysis of an association allele present in all populations indicates that selection is a significant factor in the spread and/or maintenance of at least some of retroelement-gene associations in D. melanogaster.

Evidence for the contribution of LTR retrotransposons to C. elegans gene evolution.

LTR retrotransposons may be important contributors to host gene evolution because they contain regulatory and coding signals. In an effort to assess the possible contribution of LTR retrotransposons to C. elegans gene evolution, we searched upstream and downstream of LTR retrotransposon sequences for the presence of predicted genes. Sixty-three percent of LTR retrotransposon sequences (79/124) are located within 1 kb of a gene or within gene boundaries. Most gene-retrotransposon associations were located along the chromosome arms. Our results are consistent with the hypothesis that LTR retrotransposons have contributed to the structural and/or regulatory evolution of genes in C. elegans.