The genome sequence of the Turnip Sawfly, Athalia rosae (Linnaeus, 1758)

We present a genome assembly from an individual female Athalia rosae (the Turnip Sawfly; Arhropoda; Insecta; Hymenoptera; Athaliidae). The genome sequence is 172 megabases in span. Most of the assembly is scaffolded into eight chromosomal pseudomolecules. The mitochondrial genome has also been assembled and is 16.3 kilobases in length. Gene annotation of this assembly on Ensembl identified 11,393 protein coding genes.


Background
Athalia rosae is a small (6 to 8 mm), orange sawfly marked with black on the head, antennae, mesonotum, tibiae, and tarsi.It is commonly referred to in Britain as the Turnip Sawfly, a reference to its importance as a pest of turnip, mustard, and other crops throughout temperate and subtropical zones of Europe, Asia, and Africa.In Britain, it was a serious pest during the 18th and early 19th century before almost disappearing until the 1940s, since when it has become common in England and Wales and occurring in smaller numbers throughout Scotland (Benson, 1952).The species breeds in Britain, but its numbers are boosted by large migrations from the near continent.Adults are on the wing from April to October. A. rosae is easily distinguished from other Athalia species from the presence of a distinctive orange and black chequerboard pattern on the mesonotum (Benson, 1952).Conversely, the dark blue-grey larvae can be difficult to separate from other species within the genus.The larvae feed on a broad range of brassicas including Armoracia rusticana (Horseradish), Barbarea vulgaris (Winter-cress), Brassica juncea (Chinese Mustard), Brassica napus (Rape), Brassica nigra (Black Mustard), and Brassica rapa (Turnip) (Liston, 1995).The species is multivoltine.
Athalia has for a long time been recognised as an ancient and distinct genus within the Tenthredinidae (Cameron, 1882).Recent research comparing sawfly mitochondrial genomes (Niu et al., 2022) has concluded that the genus Athalia is within a newly defined family, Athaliidae, separate from the Tenthredinidae.The chromosomal genome of Athalia rosae will help with research into the diversification and contribute insight into phylogeographic distribution pattern of this group of sawflies across their Eurasian and African range (Niu et al., 2022).

Genome sequence report
The genome was sequenced from one female Athalia rosae (Figure 1) collected from Wytham Woods, UK (latitude 51.77, longitude -1.34).A total of 103-fold coverage in Pacific Biosciences single-molecule HiFi long reads and 205-fold coverage in 10X Genomics read clouds were generated.Primary assembly contigs were scaffolded with chromosome conformation Hi-C data.Manual assembly curation corrected 39 missing or mis-joins and removed five haplotypic duplications, reducing the scaffold number by 72.55%, and increasing the scaffold N50 by 90%.
The final assembly has a total length of 172.0 Mb in 14 sequence scaffolds with a scaffold N50 of 25.5 Mb (Table 1).Most (99.4%) of the assembly sequence was assigned to eight chromosomal-level scaffolds.Chromosome-scale scaffolds confirmed by the Hi-C data are named in order of size (Figure 2-Figure 5; Table 2).The assembly has a BUSCO v5.3.2 (Manni et al., 2021) completeness of 95.5% (single 95.3%, duplicated 0.2%) using the hymenoptera_odb10 reference set.While not fully phased, the assembly deposited is of one haplotype.Contigs corresponding to the second haplotype have also been deposited.

Genome annotation report
Annotation of the GCA_917208135.1 assembly was generated using the Ensembl genome annotation pipeline (Table 1; https://rapid.ensembl.org/Athalia_rosae_GCA_917208135.1/).The resulting annotation includes 11,393 protein coding genes with an average length of 8,560.96and an average coding length of 1,651.72,and 2,142 non-protein coding genes.There is an average of 6.78 exons and 5.78 introns per canonical protein coding transcript, with an average intron length of 916.89.

Sample acquisition and nucleic acid extraction
One Athalia rosae (iyAthRosa1) specimen was collected by netting in Wytham Woods, Oxfordshire (biological vice-county: Berkshire), UK (latitude 51.77, longitude -1.34) on 28 August 2019.The specimen was collected and identified by Liam Crowley (University of Oxford) and snap-frozen on dry ice.This specimen was used for DNA sequencing.
A second specimen (iyAthRosa6) was collected using by netting in Hartslock Reserve, Oxfordshire, UK (latitude 51.51, longitude -1.11) on 20 August 2020.This specimen was collected and identified by Gavin Broad (Natural History Museum) and snap-frozen on dry ice.This specimen was used for RNA-Seq (iyAthRosa6).
DNA was extracted at the Tree of Life laboratory, Wellcome Sanger Institute (WSI).The iyAthRosa1 sample was weighed and dissected on dry ice with tissue set aside for Hi-C   sequencing.Thorax tissue was disrupted using a Nippi Powermasher fitted with a BioMasher pestle.High molecular weight (HMW) DNA was extracted using the Qiagen MagAttract HMW DNA extraction kit.Low molecular weight DNA was removed from a 20 ng aliquot of extracted DNA using 0.8X AMpure XP purification kit prior to 10X Chromium sequencing; a minimum of 50 ng DNA was submitted for 10X sequencing.
HMW DNA was sheared into an average fragment size of 12-20 kb in a Megaruptor 3 system with speed setting 30.Sheared DNA was purified by solid-phase reversible immobilisation using AMPure PB beads with a 1.8X ratio of beads to sample to remove the shorter fragments and concentrate the DNA sample.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.
RNA was extracted from whole organism tissue of iyAthRosa6 in the Tree of Life Laboratory at the WSI using TRIzol, according to the manufacturer's instructions.RNA was then eluted in 50 μl RNAse-free water and its concentration assessed using a Nanodrop spectrophotometer and Qubit Fluorometer using the Qubit RNA Broad-Range (BR) Assay kit.Analysis of the integrity of the RNA was done using Agilent RNA 6000 Pico Kit and Eukaryotic Total RNA assay.

Sequencing
Pacific Biosciences HiFi circular consensus and 10X Genomics read cloud DNA sequencing libraries were constructed according to the manufacturers' instructions.Poly(A)    3 contains a list of all software tool versions used, where appropriate.

Genome annotation
The Ensembl gene annotation system (Aken et al., 2016) was used to generate annotation for the A. rosae assembly (GCA_917208135.1).Annotation was created primarily through alignment of transcriptomic data to the genome, with gap filling via protein to-genome alignments of a select set of proteins from UniProt (UniProt Consortium, 2019).I find that the level of detail provided for the sample acquisition, extraction and sequencing is thorough.However, this manuscript does not reference in enough detail the bioinformatic methods used to allow replication by others.For example, it is unclear whether the sequenced reads were preprocessed, and the type of parameters used in the different steps reported.Of course, it is not in the scope of this manuscript to provide a particularly detailed view of bioinformatic analyses performed by DToL, but I would expect DToL to publish an explanation of its bioinformatic pipelines for individual manuscripts to reference.

Minor points:
It should be made explicit that BUSCO was made using the genome mode (not the transcriptome or protein mode), so that it is unambiguous that the completeness values reported do reflect the quality of the assembly and not the quality of the annotation.(There would be value in reporting the completeness of the annotation, but this is not essential for a data report).
"average length of 8,560.96and an average coding length of 1,651.72".I would include a unit (nucleotides) and explicitly say whether or not the length of 8,560.96includes introns or not.
"but its numbers are boosted by large migrations from the near continent".Using the term "mainland Europe" is more clear that "the near continent".
What is dataset CAKJPL01 referenced in Fig. 2?
Is the rationale for creating the dataset(s) clearly described?

Shu-Jun Wei
Beijing Academy of Agriculture and Forestry Sciences, Beijing, China This study reported the chromosome-level genome for the turnip sawfly (Hymenoptera; Athaliidae) using Pacific Biosciences single-molecule HiFi long reads, 10X Genomics, and Hi-C technologies.This genome assembly has a BUSCO completeness of 95.5%.The results will help understand the phylogeographic distribution of sawflies and the evolution of Hymenoptera.Here are some points for clarification: Are there any voucher specimens or DNA available?1.
Can you provide information about the completeness of the annotated protein-coding genes?

2.
Was the mitochondrial genome annotated?Please include the annotation results for the mitochondrial genome.

Is the rationale for creating the dataset(s) clearly described? Yes
Are the protocols appropriate and is the work technically sound?

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.
Reviewer Expertise: Population genetics, genomics, and pest control.
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.

Xiaohan Shu
Zhejiang University, Sanya, China This article describes the genome sequencing and annotation of the Turnip Sawfly (Athalia rosae).The genome of Athalia rosae is a good resource and useful for researchers studying sawfly.Overall this genome is high quality and the manuscript provides a sufficient description of the methods used to generate the genome.

Are the datasets clearly presented in a useable and accessible format? Yes
Competing Interests: No competing interests were disclosed.
Reviewer Expertise: insect genomics, ecology & evolution 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.

Sandro Patroca da Silva
Evandro Chagas Institute, Ananindeua, Brazil The article entitled "The genome sequence of the Turnip Sawfly, Athalia rosae (Linnaeus, 1758)" describes the sequencing of an insect species using different methodologies and sequencing platforms, as well as bioinformatics methodologies to obtain the nuclear and mitochondrial genome of the Turnip Sawfly.In my opinion, the laboratory methodologies and computational analysis tools are appropriate, and I did not observe any issues with their utilization.

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.
Reviewer Expertise: I am a virologist and have expertise in NGS technology and bioinformatic analysis.At the moment, I am beginning to work with the mitochondrial genome of arthropods.
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.

Figure 2 .
Figure 2. Genome assembly of Athalia rosae, iyAthRosa1.1: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 171,971,399 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 (32,626,317 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (25,530,711 and 16,594,773 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 hymenoptera_odb10 set is shown in the top right.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/iyAthRosa1.1/dataset/ CAKJPL01/snail.

Figure 3 .
Figure 3. Genome assembly of Athalia rosae, iyAthRosa1.1:GC coverage.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/iyAthRosa1.1/dataset/CAKJPL01/blob.

Reviewer
Report 14 July 2023 https://doi.org/10.21956/wellcomeopenres.21057.r58697© 2023 da Silva S. 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.

Table 3 . Software tools and versions used.
Ethics and compliance issuesThe 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.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.All efforts are undertaken to minimise the suffering of animals used for sequencing.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.

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? Partly Are the datasets clearly presented in a useable and accessible format? Yes Competing Interests:
No competing interests were disclosed.

have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.
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.