The genome sequence of the Small Square-spot, Diarsia rubi (Vieweg, 1790)

We present a genome assembly from an individual female Diarsia rubi (the Small Square-spot; Arthropoda; Insecta; Lepidoptera; Noctuidae). The genome sequence is 624.9 megabases in span. Most of the assembly is scaffolded into 32 chromosomal pseudomolecules, including the Z and W sex chromosomes. The mitochondrial genome has also been assembled and is 15.3 kilobases in length. Gene annotation of this assembly on Ensembl identified 19,173 protein coding genes.


Background
The genus Diarsia contains around 100 species of noctuid moths, found primarily across the Palaearctic, Indomalayan and Australasian realms; many Diarsia species have similar wing markings (Gyulai & Saldaitis, 2019).Even within a region with low diversity in the genus, such as Britain and Ireland with just four or five species, it can sometimes be difficult to distinguish species using external characters alone.The Small Square-spot D. rubi is a common and often abundant moth found across England, Wales, Scotland, Northern Ireland and Ireland (Randle et al., 2019).The species is also common across most of Europe, with sporadic records from further east to Russia and Tajikistan (GBIF Secretariat, 2022).The moth is frequent in gardens, woodlands and grassland habitats with the polyphagous larvae feeding on a wide range of herbaceous plants (Skinner & Wilson, 2009;South, 1961).
The common name derives a square-shaped russet brown patch between the pale orbicular and reniform stigmata (oval and kidney marks), on a grey-brown ground colour.The same pattern is also present in D. mendica, which may be distinguished from D. rubi by subtle wing pattern differences and genitalia morphology (Townsend et al., 2010).D. florida lacks clear genitalic differences from D. rubi, and further work is needed to clarify if these are distinct species (Skinner & Wilson, 2009;Townsend et al., 2010).
The life cycle of D. rubi reveals an interesting case of environmental influence on development.Although the moth is single brooded in the north of its range including Scotland, further south across England and Wales the species has two generations each year that differ in appearance.Moths from the spring generation, on the wing in May and June, are larger and generally paler than moths from the summer generation, on the wing in August and September (Skinner & Wilson, 2009;South, 1961).It is unclear if this is a consequence of diet, since the larvae giving rise to the spring emergence feed for longer (Ford, 1967), or an environmental polymorphism triggered by a variable such as day length, analogous to the comma butterfly Nymphalis c-album see (Nylin, 1989).
A complete genome sequence from D. rubi will facilitate research into adaptations to polyphagy, and the interaction between genes and environment.

Genome sequence report
The genome was sequenced from one female Diarsia rubi specimen (Figure 1) collected from Wytham Woods, Oxfordshire, UK (latitude 51.77, longitude -1.34).A total of 33-fold coverage in Pacific Biosciences single-molecule HiFi long reads and 52-fold coverage in 10X Genomics read clouds was generated.Primary assembly contigs were scaffolded with chromosome conformation Hi-C data.Manual assembly curation corrected 10 missing joins or mis-joins, reducing the scaffold number by 22.73%.
The final assembly has a total length of 624.9 Mb in 34 sequence scaffolds with a scaffold N50 of 21.1 Mb (Table 1).Most (99.99%) of the assembly sequence was assigned to 32 chromosomal-level scaffolds, representing 30 autosomes, and the Z and W sex chromosomes.Chromosome-scale scaffolds confirmed by the Hi-C data are named in order of size (Figure 2-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.
The estimated Quality Value (QV) of the final assembly is 61.2 with k-mer completeness of 100%, and the assembly has a BUSCO v5.3.2 completeness of 98.9% (single 98.4%, duplicated 0.4%) using the lepidoptera_odb10 reference set (n = 5,286).
Metadata for specimens, spectral estimates, sequencing runs, contaminants and pre-curation assembly statistics can be found at https://links.tol.sanger.ac.uk/species/987925.
The resulting annotation includes 19,371 transcribed mRNAs from 19,173 protein-coding genes.

Sample acquisition and nucleic acid extraction
A female Diarsia rubi (ilDiaRubi1) was collected from Wytham Woods, Oxfordshire (biological vice-county: Berkshire), UK (latitude 51.77, longitude -1.34) on 8 September 2020.The  specimen was taken from woodland habitat by Douglas Boyes (University of Oxford) using a light trap.The specimen was identified by the collector and snap-frozen on dry ice.
DNA was extracted at the Tree of Life laboratory, Wellcome Sanger Institute (WSI).The ilDiaRubi1 sample was weighed and dissected on dry ice with head tissue set aside for Hi-C sequencing.Thorax tissue was cryogenically disrupted to a fine powder using a Covaris cryoPREP Automated Dry Pul-veriser, receiving multiple impacts.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 the 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.

Sequencing
Pacific Biosciences HiFi circular consensus and 10X Genomics read cloud DNA sequencing libraries were constructed according to the manufacturers' instructions.DNA sequencing was performed by the Scientific Operations core at the WSI on Pacific Biosciences SEQUEL II (HiFi) and Illumina NovaSeq 6000 (10X) instruments.Hi-C data were also generated from head tissue of ilDiaRubi1 using the Arima v2 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).One round of polishing was performed by aligning 10X Genomics read data to the assembly with Long Ranger ALIGN, calling variants with FreeBayes (Garrison & Marth, 2012).The assembly was then scaffolded with Hi-C data (Rao et al., 2014) using YaHS (Zhou et al., 2023).The assembly was checked for as described previously (Howe et al., 2021).Manual curation was performed using gHiGlass (Kerpedjiev et al., 2018) and Pretext (Harry, 2022).The mitochondrial genome was assembled using MitoHiFi (Uliano-Silva et al., 2022), 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.To evaluate the assembly, MerquryFK was used to estimate consensus quality (QV) scores and k-mer completeness (Rhie et al., 2020).The genome was analysed within the BlobToolKit environment (Challis et al., 2020) andBUSCO scores (Manni et al., 2021;Simão et al., 2015) were calculated.Table 3 contains a list of software tool versions and sources.

Genome annotation
The BRAKER2 pipeline (Brůna et al., 2021) was used in the default protein mode to generate annotation for the Diarsia rubi assembly (GCA_932274075.1) in Ensembl Rapid Release.

Ethics and compliance issues
The materials that have contributed to this genome

Paula Escuer
University of Neuchâtel, Neuchâtel, Switzerland In this research article is presented the genome sequence of a female individual of Diarsia rubi (the Small Square-spot; Arthropoda; Insecta; Lepidoptera; Noctuidae).The assembly size contains 624.9 Mb condensed in 34 scaffolds with a scaffold N50 of 21.1 Mb (Table 1, Figure 5).The 99.9% of the assembly is distributed across 32 chromosomal level scaffolds, which represents the 30 autosomes plus the Z and W sex chromosomes.The mitochondrial genome is available and comprises 15.3 kb of length.The BUSCO pipeline identified 98.9% of complete genes, indicating a high completeness of the genome (Table 3, Figure 2).Finally, they identified 19,173 protein-coding genes using the default protein mode of the annotation pipeline BRAKER.This high quality genome will help to resolve questions about adaptations to their characteristic polyphagy and the evolution of Lepidoptera.
The article is clear and well written, they used standard protocols and pipelines for the sequencing and to assembly the genome.The resulting dataset presents good quality statistics, high contiguity and completeness, I recommend using RNA-seq data to improve the quality of annotation if possible.

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: Biodiversity, Genomics, Speciation, Genome assembly and annotation 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 Diarsia rubi, ilDiaRubi1.1: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 624,929,271 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 (32,634,975 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (21,075,383 and 13,933,945 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 lepidoptera_odb10 set is shown in the top right.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/ilDiaRubi1.1/dataset/ CAKNZU01.1/snail.
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

Figure 5 .
Figure 5. Genome assembly of Diarsia rubi, ilDiaRubi1.1:Hi-C contact map.Hi-C contact map of the ilDiaRubi1.1 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=RZTqYGymTpKj7IKceK8PxQ.

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.

have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.
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