Abstract
We present a genome assembly from an individual male Bruchidius siliquastri (the Judas tree Seed Beetle; Arthropoda; Insecta; Coleoptera; Chrysomelidae). The genome sequence is 375.6 megabases in span. Most of the assembly is scaffolded into 11 chromosomal pseudomolecules, including the X and Y sex chromosomes. The mitochondrial genome has also been assembled and is 15.81 kilobases in length. Gene annotation of this assembly on Ensembl identified 17,940 protein coding genes.
Keywords: Bruchidius siliquastri, the Judas tree Seed Beetle, genome sequence, chromosomal, Coleoptera
Species taxonomy
Eukaryota; Opisthokonta; Metazoa; Eumetazoa; Bilateria; Protostomia; Ecdysozoa; Panarthropoda; Arthropoda; Mandibulata; Pancrustacea; Hexapoda; Insecta; Dicondylia; Pterygota; Neoptera; Endopterygota; Coleoptera; Polyphaga; Cucujiformia; Chrysomeloidea; Chrysomelidae; Bruchinae; Bruchini; Bruchidius; Bruchidius siliquastri Delobel, 2007 (NCBI:txid1649775).
Background
Bruchidius siliquastri was described as new to science based on specimens collected in southern France by Delobel in Kergoat et al. (2007), who also cite specimens from Hungary and China (not included in the type series). They assume that the species was imported to Europe from China. It has since been reported from Slovakia ( Kollár et al., 2009), Belgium ( Hanssens, 2009), the Czech Republic ( Šefrova et al., 2010), Bulgaria ( Stojanova et al., 2011), Serbia ( Gavrilović & Savić, 2013), Germany ( Rheinheimer & Hassler, 2013), Turkey ( Hizal & Parlak, 2013), Ukraine ( Martynov & Nikulina, 2015), Italy ( Yus Ramos & Bocci, 2017), Romania ( Pintilioaie et al., 2018), Korea ( Jeong et al., 2022), as well as from Spain, where its life history and immature stages have been explored ( Yus Ramos et al., 2009a; Yus Ramos et al., 2009b; Yus Ramos et al., 2009c). It was first reported in Britain from South Kensington in 2014 ( Barclay, 2014) and has since become widespread in the London area and in other cities and towns in southern Britain. Although Britain is the northern limit of its currently known distribution, the beetle is actively expanding its range, and trees where it was apparently absent 5 to 10 years ago now support populations.
B. siliquastri develops in the seed pods of leguminous trees of the genus Cercis (Fabaceae), particularly the Judas tree Cercis siliquastrum Linnaeus. Cercis occurs throughout the northern hemisphere, and while the Judas tree is native to southern Europe and western Asia, it is a popular planted ornamental in cities and parks elsewhere because of its bright flowers and attractive foliage. It is common in the Levant, and may owe its name to a legend that Judas Iscariot hanged himself from one ( Barclay, 2014), but the majority of London specimens are much too small for this. An alternative theory is that the name is a corruption of ‘Judea’, the apparent origin of European specimens. The tree features in Middle Eastern art and poetry, and the edible flowers and young leaves are sometimes used as a culinary garnish, but there is no indication that the beetle was present in Europe or Western Asia until the 2000s.
Adults of B. siliquastri are most easily collected by beating flowers, pods and foliage of Cercis from May to August, but they can also be reared from pods collected in spring. Stojanova et al. (2011) observed a large parasitoid fauna associated with Bruchidius siliquastri in Bulgaria, but no species of Hymenoptera have been reared from the London population of this beetle so far. The only other herbivorous insect recorded feeding on the trees was the jumping plant louse Cacopsylla pulchella (Löw) (Hemiptera: Psyllidae).
Bruchidius siliquastri can immediately be recognised from nearly all other species of Bruchinae occurring in Northern Europe by its bright red abdomen. This is conspicuous when the beetle is on its back, but is also visible dorsally, since the red pygidium extends well beyond the apices of the elytra. Apart from this striking feature it resembles a slightly small example of the common Bruchidius villosus (Fabricius), being black with whitish pubescence on the dorsal surface. The only other European Bruchinae with a reddish abdomen is the cosmopolitan Acanthoscelides obtectus (Say), an occasional kitchen and warehouse pest associated with dried beans. Acanthoscelides can resemble a large Bruchidius when stored in ethanol, but immediately differs by the dense brownish vestiture of elytra and pronotum when alive or dry. It is also immediately recognisable by the sharp teeth on the hind femora, along with its broader shape ( Duff, 2016).
The specimens used for the Darwin Tree of Life project were collected on 23 July 2021 by a small Natural History Museum team, conducting a survey of Wetherby Gardens at the request of Natural History Museum patrons Holly Smith and Neil Osborn. The beetles were beaten in large numbers from a small Judas Tree growing from the pavement near the entrance to Wetherby Garden Square, less than a mile from the original South Kensington locality ( Barclay, 2014).
Genome sequence report
The genome was sequenced from one male Bruchidius siliquastri ( Figure 1) collected from Wetherby Gardens, Kensington, London, UK (51.49, –0.18). A total of 57-fold coverage in Pacific Biosciences single-molecule HiFi long reads was generated. Primary assembly contigs were scaffolded with chromosome conformation Hi-C data. Manual assembly curation corrected 26 missing joins or mis-joins and removed 6 haplotypic duplications, reducing the assembly length by 1.11% and the scaffold number by 44.83%, and increasing the scaffold N50 by 10.13%.
Figure 1.
Photographs of Bruchidius siliquastri: a) A specimen collected in Kensington in 2014: photograph by Harry Taylor (Natural History Museum); b)– d) The specimen used for genome sequencing (icBruSili1): b) dorsal view, c) lateral view and d) ventral view.
The final assembly has a total length of 375.6 Mb in 15 sequence scaffolds with a scaffold N50 of 39.9 Mb ( Table 1). The snailplot in Figure 2 provides a summary of the assembly statistics, while the distribution of assembly scaffolds on GC proportion and coverage is shown in Figure 3. The cumulative assembly plot in Figure 4 shows curves for subsets of scaffolds assigned to different phyla. Most (99.95%) of the assembly sequence was assigned to 11 chromosomal-level scaffolds, representing 9 autosomes and the X and Y sex chromosomes. Chromosome-scale scaffolds confirmed by the Hi-C data are named in order of size ( Figure 5; Table 2). While not fully phased, the assembly deposited is of one haplotype. Contigs corresponding to the second haplotype have also been deposited. The mitochondrial genome was also assembled and can be found as a contig within the multifasta file of the genome submission.
Figure 2. Genome assembly of Bruchidius siliquastri, icBruSili1.1: metrics.
The BlobToolKit Snailplot shows N50 metrics and BUSCO gene completeness. The main plot is divided into 1,000 size-ordered bins around the circumference with each bin representing 0.1% of the 375,583,928 bp assembly. The distribution of scaffold lengths is shown in dark grey with the plot radius scaled to the longest scaffold present in the assembly (49,182,353 bp, shown in red). Orange and pale-orange arcs show the N50 and N90 scaffold lengths (39,857,073 and 26,473,722 bp), respectively. The pale grey spiral shows the cumulative scaffold count on a log scale with white scale lines showing successive orders of magnitude. The blue and pale-blue area around the outside of the plot shows the distribution of GC, AT and N percentages in the same bins as the inner plot. A summary of complete, fragmented, duplicated and missing BUSCO genes in the endopterygota_odb10 set is shown in the top right. An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/CASGFI01/dataset/CASGFI01/snail.
Figure 3. Genome assembly of Bruchidius siliquastri, icBruSili1.1: BlobToolKit GC-coverage plot.
Scaffolds are coloured by phylum. Circles are sized in proportion to scaffold length. Histograms show the distribution of scaffold length sum along each axis. An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/CASGFI01/dataset/CASGFI01/blob.
Figure 4. Genome assembly of Bruchidius siliquastri, icBruSili1.1: BlobToolKit cumulative sequence plot.
The grey line shows cumulative length for all scaffolds. Coloured lines show cumulative lengths of scaffolds assigned to each phylum using the buscogenes taxrule. An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/CASGFI01/dataset/CASGFI01/cumulative.
Figure 5. Genome assembly of Bruchidius siliquastri, icBruSili1.1: Hi-C contact map of the icBruSili1.1 assembly, visualised using HiGlass.
Chromosomes are shown in order of size from left to right and top to bottom. An interactive version of this figure may be viewed at https://genome-note-higlass.tol.sanger.ac.uk/l/?d=ff3I2oGrSVaZWymS1fyS7w.
Table 1. Genome data for Bruchidius siliquastri, icBruSili1.1.
| Project accession data | ||
|---|---|---|
| Assembly identifier | icBruSili1.1 | |
| Species | Bruchidius siliquastri | |
| Specimen | icBruSili1 | |
| NCBI taxonomy ID | 1649775 | |
| BioProject | PRJEB59372 | |
| BioSample ID | SAMEA111458828 | |
| Isolate information | icBruSili1, male: abdomen (DNA sequencing); head and thorax
(Hi-C sequencing) |
|
| Assembly metrics * | Benchmark | |
| Consensus quality (QV) | 60.3 | ≥ 50 |
| k-mer completeness | 100.0% | ≥ 95% |
| BUSCO ** | C:97.6%[S:97.4%,D:0.3%],
F:0.4%,M:1.9%,n:2,124 |
C ≥ 95% |
| Percentage of assembly mapped to
chromosomes |
99.95% | ≥ 95% |
| Sex chromosomes | XY | localised homologous pairs |
| Organelles | Mitochondrial genome: 15.81 kb | complete single alleles |
| Raw data accessions | ||
| PacificBiosciences SEQUEL II | ERR10841317 | |
| Hi-C Illumina | ERR10851510 | |
| Genome assembly | ||
| Assembly accession | GCA_949316355.1 | |
| Accession of alternate haplotype | GCA_949316485.1 | |
| Span (Mb) | 375.6 | |
| Number of contigs | 87 | |
| Contig N50 length (Mb) | 7.0 | |
| Number of scaffolds | 15 | |
| Scaffold N50 length (Mb) | 39.9 | |
| Longest scaffold (Mb) | 49.18 | |
| Genome annotation | ||
| Number of protein-coding genes | 17,940 | |
| Number of gene transcripts | 18,096 | |
* Assembly metric benchmarks are adapted from column VGP-2020 of “Table 1: Proposed standards and metrics for defining genome assembly quality” from ( Rhie et al., 2021).
** BUSCO scores based on the endopterygota_odb10 BUSCO set using version 5.3.2. C = complete [S = single copy, D = duplicated], F = fragmented, M = missing, n = number of orthologues in comparison. A full set of BUSCO scores is available at https://blobtoolkit.genomehubs.org/view/CASGFI01/dataset/CASGFI01/busco.
Table 2. Chromosomal pseudomolecules in the genome assembly of Bruchidius siliquastri, icBruSili1.
| INSDC accession | Chromosome | Length (Mb) | GC% |
|---|---|---|---|
| OX438529.1 | 1 | 49.18 | 36.0 |
| OX438530.1 | 2 | 45.38 | 36.5 |
| OX438531.1 | 3 | 42.04 | 36.5 |
| OX438532.1 | 4 | 41.56 | 36.5 |
| OX438533.1 | 5 | 39.86 | 36.5 |
| OX438534.1 | 6 | 39.04 | 36.5 |
| OX438535.1 | 7 | 36.82 | 36.5 |
| OX438536.1 | 8 | 28.49 | 37.0 |
| OX438537.1 | 9 | 26.47 | 37.0 |
| OX438538.1 | X | 22.91 | 37.0 |
| OX438539.1 | Y | 3.5 | 36.5 |
| OX438540.1 | MT | 0.02 | 23.0 |
The estimated Quality Value (QV) of the final assembly is 60.3 with k-mer completeness of 100.0%, and the assembly has a BUSCO v5.3.2 completeness of 97.6% (single = 97.4%, duplicated = 0.3%), using the endopterygota_odb10 reference set ( n = 2,124).
Metadata for specimens, barcode results, spectra estimates, sequencing runs, contaminants and pre-curation assembly statistics are given at https://links.tol.sanger.ac.uk/species/1649775.
Genome annotation report
The Bruchidius siliquastri genome assembly (GCA_949316355.1) was annotated using the Ensembl rapid annotation pipeline at the European Bioinformatics Institute (EBI). The resulting annotation includes 18,096 transcribed mRNAs from 17,940 protein-coding genes ( Table 1; https://rapid.ensembl.org/Bruchidius_siliquastri_GCA_949316355.1/Info/Index)..
Methods
Sample acquisition and nucleic acid extraction
A male Bruchidius siliquastri (specimen ID NHMUK014440509, ToLID icBruSili1) was handpicked from Wetherby Gardens, Kensington, London, UK (latitude 51.49, longitude –0.18) on 2021-07-23. The specimen was collected by Maxwell Barclay, Michael Geiser, Keita Matsumoto and Emily Pash (Natural History Museum) and identified by Maxwell Barclay, and then preserved by dry freezing at –80 °C.
The workflow for high molecular weight (HMW) DNA extraction at the WSI includes a sequence of core procedures: sample preparation; sample homogenisation, DNA extraction, fragmentation, and clean-up. In sample preparation, the icBruSili1 sample was weighed and dissected on dry ice ( Jay et al., 2023). Tissue from the abdomen was homogenised using a PowerMasher II tissue disruptor ( Denton et al., 2023a). HMW DNA was extracted in the WSI Scientific Operations core using the Automated MagAttract v2 protocol ( Oatley et al., 2023). HMW DNA was sheared into an average fragment size of 12–20 kb in a Megaruptor 3 system with speed setting 31 ( Bates et al., 2023). Sheared DNA was purified by solid-phase reversible immobilisation ( Strickland et al., 2023): in brief, the method employs a 1.8X ratio of AMPure PB beads to sample to eliminate shorter fragments and concentrate the DNA. The concentration of the sheared and purified DNA was assessed using a Nanodrop spectrophotometer and Qubit Fluorometer and Qubit dsDNA High Sensitivity Assay kit. Fragment size distribution was evaluated by running the sample on the FemtoPulse system.
Protocols developed by the Wellcome Sanger Institute (WSI) Tree of Life core laboratory have been deposited on protocols.io ( Denton et al., 2023b).
Sequencing
Pacific Biosciences HiFi circular consensus DNA sequencing libraries were constructed according to the manufacturers’ instructions. DNA sequencing was performed by the Scientific Operations core at the WSI on a Pacific Biosciences SEQUEL II instrument. Hi-C data were also generated from head and thorax tissue of icBruSili1 using the Arima2 kit and sequenced on the Illumina NovaSeq 6000 instrument.
Genome assembly, curation and evaluation
Assembly was carried out with Hifiasm ( Cheng et al., 2021) and haplotypic duplication was identified and removed with purge_dups ( Guan et al., 2020). The assembly was then scaffolded with Hi-C data ( Rao et al., 2014) using YaHS ( Zhou et al., 2023). The assembly was checked for contamination and corrected as described previously ( Howe et al., 2021). Manual curation was performed using HiGlass ( Kerpedjiev et al., 2018) and Pretext ( Harry, 2022). The mitochondrial genome was assembled using MitoHiFi ( Uliano-Silva et al., 2023), which runs MitoFinder ( Allio et al., 2020) or MITOS ( Bernt et al., 2013) and uses these annotations to select the final mitochondrial contig and to ensure the general quality of the sequence.
A Hi-C map for the final assembly was produced using bwa-mem2 ( Vasimuddin et al., 2019) in the Cooler file format ( Abdennur & Mirny, 2020). To assess the assembly metrics, the k-mer completeness and QV consensus quality values were calculated in Merqury ( Rhie et al., 2020). This work was done using Nextflow ( Di Tommaso et al., 2017) DSL2 pipelines “sanger-tol/readmapping” ( Surana et al., 2023a) and “sanger-tol/genomenote” ( Surana et al., 2023b). The genome was analysed within the BlobToolKit environment ( Challis et al., 2020) and BUSCO scores ( Manni et al., 2021; Simão et al., 2015) were calculated.
Table 3 contains a list of relevant software tool versions and sources.
Table 3. Software tools: versions and sources.
| Software tool | Version | Source |
|---|---|---|
| BlobToolKit | 4.1.7 | https://github.com/blobtoolkit/blobtoolkit |
| BUSCO | 5.3.2 | https://gitlab.com/ezlab/busco |
| Hifiasm | 0.16.1-r375 | https://github.com/chhylp123/hifiasm |
| HiGlass | 1.11.6 | https://github.com/higlass/higlass |
| Merqury | MerquryFK |
https://github.com/thegenemyers/MERQURY.
FK |
| MitoHiFi | 2 | https://github.com/marcelauliano/MitoHiFi |
| PretextView | 0.2 | https://github.com/wtsi-hpag/PretextView |
| purge_dups | 1.2.3 | https://github.com/dfguan/purge_dups |
| sanger-tol/
genomenote |
v1.0 | https://github.com/sanger-tol/genomenote |
| sanger-tol/
readmapping |
1.1.0 |
https://github.com/sanger-tol/readmapping/
tree/1.1.0 |
| YaHS | 1.2a | https://github.com/c-zhou/yahs |
Genome annotation
The BRAKER2 pipeline ( Brůna et al., 2021) was used in the default protein mode to generate annotation for the Bruchidius siliquastri assembly (GCA_949316355.1) in Ensembl Rapid Release at the EBI.
Wellcome Sanger Institute – Legal and Governance
The materials that have contributed to this genome note have been supplied by a Darwin Tree of Life Partner. The submission of materials by a Darwin Tree of Life Partner is subject to the ‘Darwin Tree of Life Project Sampling Code of Practice’, which can be found in full on the Darwin Tree of Life website here. By agreeing with and signing up to the Sampling Code of Practice, the Darwin Tree of Life Partner agrees they will meet the legal and ethical requirements and standards set out within this document in respect of all samples acquired for, and supplied to, the Darwin Tree of Life Project.
Further, the Wellcome Sanger Institute employs a process whereby due diligence is carried out proportionate to the nature of the materials themselves, and the circumstances under which they have been/are to be collected and provided for use. The purpose of this is to address and mitigate any potential legal and/or ethical implications of receipt and use of the materials as part of the research project, and to ensure that in doing so we align with best practice wherever possible. The overarching areas of consideration are:
• Ethical review of provenance and sourcing of the material
• Legality of collection, transfer and use (national and international)
Each transfer of samples is further undertaken according to a Research Collaboration Agreement or Material Transfer Agreement entered into by the Darwin Tree of Life Partner, Genome Research Limited (operating as the Wellcome Sanger Institute), and in some circumstances other Darwin Tree of Life collaborators.
Funding Statement
This work was supported by Wellcome through core funding to the Wellcome Sanger Institute [206194, <a href=https://doi.org/10.35802/206194>https://doi.org/10.35802/206194</a>] and the Darwin Tree of Life Discretionary Award [218328, <a href=https://doi.org/10.35802/218328>https://doi.org/10.35802/218328 </a>].
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
[version 1; peer review: 2 approved]
Data availability
European Nucleotide Archive: Bruchidius siliquastri. Accession number PRJEB59372; https://identifiers.org/ena.embl/PRJEB59372 ( Wellcome Sanger Institute, 2023). The genome sequence is released openly for reuse. The Bruchidius siliquastri genome sequencing initiative is part of the Darwin Tree of Life (DToL) project. All raw sequence data and the assembly have been deposited in INSDC databases. Raw data and assembly accession identifiers are reported in Table 1.
Author information
Members of the Natural History Museum Genome Acquisition Lab are listed here: https://doi.org/10.5281/zenodo.7139035.
Members of the Darwin Tree of Life Barcoding collective are listed here: https://doi.org/10.5281/zenodo.4893703.
Members of the Wellcome Sanger Institute Tree of Life Management, Samples and Laboratory team are listed here: https://doi.org/10.5281/zenodo.10066175.
Members of Wellcome Sanger Institute Scientific Operations: Sequencing Operations are listed here: https://doi.org/10.5281/zenodo.10043364.
Members of the Wellcome Sanger Institute Tree of Life Core Informatics team are listed here: https://doi.org/10.5281/zenodo.10066637.
Members of the Tree of Life Core Informatics collective are listed here: https://doi.org/10.5281/zenodo.5013541.
Members of the Darwin Tree of Life Consortium are listed here: https://doi.org/10.5281/zenodo.4783558.
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