The genome sequence of the giant willow aphid, Tuberolachnus salignus (Gmelin, 1790)

We present a genome assembly from an individual female Tuberolachnus salignus (the giant willow aphid; Arthropoda; Insecta; Hemiptera; Aphididae). The genome sequence is 456.8 megabases in span. Most of the assembly is scaffolded into 10 chromosomal pseudomolecules. The mitochondrial genome has also been assembled and is 22.43 kilobases in length.


Background
The giant willow aphid, Tuberolachnus salignus (Gmelin, 1790), is one of the largest aphids (5 mm), and is easily identifiable due to its large size and the conical tubercle on the dorsum (Blackman & Eastpop, 1994).Their bodies are black in colour with a mesh-like grey covering which gives them a uniformly spotty appearance.Their legs are red and black.
Giant willow aphids feed on willow species (Blackman & Eastpop, 1994).In the UK, willow is used as a biomass crop, and the damage caused by the aphids reduces yield (Aradottir et al., 2012).Further, wild Bees feed on the honeydew they produce, which has been shown to reduce the success of bee activity, reducing pollination in important crops (Sopow et al., 2017).Therefore, gaining an understanding of the giant willow aphid is important to enable management strategies to protect important crops.
Giant willow aphids reproduce and feed on a single host and are presumed to only reproduce parthenogenically as no males have ever been found (Blackman & Eastpop, 1994;Sopow et al., 2017).Very few individuals have been recorded in the spring and it remains unclear where they go or if an alternative host is used during this period.Recently, colonies were found feeding on quince, Cydonia oblonga Mill., in May (Salisbury, 2022).They are thought to have originated in Asia, but now have a worldwide distribution, predominantly found in Europe, including the UK and Ireland.More recently, they have been discovered in Australia and New Zealand (Sopow et al., 2017).Perhaps due to their asexual reproduction, there is little genetic diversity between different populations; a study on 27 populations in 5 countries, found only 16 genotypes (Aradottir et al., 2012).
The giant willow aphid's large size makes it a useful model for aphid study.It is more amenable than smaller aphids to microinjections for the study of electrophysiology, the effects of RNAi molecules, and genetic manipulation.The genome of this aphid will enable more such studies.
We present a chromosomally complete genome sequence for Tuberolachnus salignus (Gmelin, 1790), based on one female specimen from Withymead Nature Reserve as part of the Darwin Tree of Life Project.This project is a collaborative effort to sequence all named eukaryotic species in the Atlantic Archipelago of Britain and Ireland.

Genome sequence report
The genome was sequenced from one female Tuberolachnus salignus (Figure 1) collected from Withymead Nature Reserve, Oxfordshire, UK (51.54, -1.14).A total of 37-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 84 missing joins or mis-joins and removed 3 haplotypic duplications, reducing the scaffold number by 39.02%, and increasing the scaffold N50 by 7.69%.
The final assembly has a total length of 456.8 Mb in 99 sequence scaffolds with a scaffold N50 of 45.8 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 (95.29%) of the assembly sequence was assigned to 10 chromosomal-level scaffolds, representing 10 autosomes.Chromosomes 3 and 7 align to the Myzus persicae X chromosome (Mathers et al., 2021), but no male data available to confirm X chromosome.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.

Sample acquisition and nucleic acid extraction
Two female Tuberolachnus salignus was collected from Withymead Nature Reserve, Oxfordshire, UK (latitude 51.54,   Fragment size distribution was evaluated by running the sample on the FemtoPulse system.

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 tissue of the whole organism of ihTubSali2 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  Table 3 contains a list of relevant software tool versions and sources.

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:  This genome report is satisfactory but there are some grammatical typos that need to be corrected.
Background: # Third sentence, bees shouldn't be capitalized: Further, wild Bees feed on the honeydew they produce # next line, fourth sentence add genetic to specify the understanding of this aphid species.Therefore, gaining a GENETIC understanding parthenogenically misspelled # Third paragraph, sentence three, specify where the colonies were found on quince.

Methods:
# first line, it should be females and were collected: Two females Tuberolachnus salignus were collected...etc.
# next line, plural specimen and agreement: The specimens were Is the rationale for creating the dataset(s) clearly described?Yes Are the protocols appropriate and is the work technically sound?Yes

Are sufficient details of methods and materials provided to allow replication by others? Yes
Are the datasets clearly presented in a useable and accessible format?Yes Competing Interests: No competing interests were disclosed.
I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.
) and the other (specimen ID Ox002687, ToLID ihTubSali2) was used for Hi-C sequencing.Protocols developed by the Wellcome Sanger Institute (WSI) Tree of Life core laboratory have been deposited on protocols.io(Dentonet al., 2023b).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 ihTubSali1 sample was weighed and dissected on dry ice(Jay et al., 2023).Tissue from the whole organism 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.

Figure 3 .
Figure 3. Genome assembly of Tuberolachnus salignus, ihTubSali1.1: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/ihTubSali1_1/dataset/ihTubSali1_1/blob.

Figure 4 .
Figure 4. Genome assembly of Tuberolachnus salignus, ihTubSali1.1: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/ihTubSali1_1/dataset/ihTubSali1_1/cumulative.

Figure 5 .
Figure 5. Genome assembly of Tuberolachnus salignus, ihTubSali1.1:Hi-C contact map of the ihTubSali1.1 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=GyIhnLv2RNi-c1J2QqK93A.

Table 1 . Genome data for Tuberolachnus salignus, ihTubSali1.1. Project accession data
longitude -1.14) on 2022-08-12 using a sweep net.The specimen was collected by Liam Crowley and James McCulloch (University of Oxford) and identified by Liam Crowley and preserved on dry ice.One specimen was used for DNA sequencing (specimen ID Ox002686, ToLID ihTubSali1

have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.
This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
https://doi.org/10.21956/wellcomeopenres.22850.r86460© 2024 Cohen Z. Zachary Cohen Insect Control and Cotton Disease Research Unit, Southern Plains Agricultural Research Center,Agricultural Research Service, United States Department of Agriculture, College Station, TX, USA