The genome sequence of the Elbow-stripe Grass-veneer, Agriphila geniculea (Haworth, 1811)

We present a genome assembly from an individual female Agriphila geniculea (the Elbow-stripe Grass-veneer; Arthropoda; Insecta; Lepidoptera; Crambidae). The genome sequence is 781.6 megabases in span. Most of the assembly is scaffolded into 30 chromosomal pseudomolecules, including the Z and W sex chromosomes. The mitochondrial genome has also been assembled and is 15.4 kilobases in length. Gene annotation of this assembly on Ensembl identified 22,132 protein coding genes.


Background
Agriphila geniculea (Haworth, 1811) is a moth in the family Crambidae, characterised by strong elbow-like markings across the forewing. It is found commonly across Britain and Ireland, and is particularly widespread in southern Britain, becoming more local north to the Outer Hebrides; it is primarily coastal in Ireland ( In Britain, the moth is most commonly encountered in gardens and dry grasslands, and can be extremely abundant on coastal sand dunes. The larvae are known to feed on grasses, dwelling in a silken tube at the base of the stems (Goater et al., 1986;Parsons & Davis, 2018). Like other members of its genus, the eggs are presumed to be non-adhesive and deposited over the foodplant by the female in flight (Léger et al., 2019). This egg-laying behaviour of the females is shared by other members of the Crambinae subfamily whose larvae feed at the base of grasses, and is associated with modifications to the female genital morphology (Léger et al., 2019).
Adults of this species are on the wing from July to October and are attracted to light (Goater et al., 1986; Parsons & Davis, 2018). During the flight season adults are readily disturbed by day from grass, but Goater notes that bushes of young conifers are also a favoured daytime resting place for the adult (Goater et al., 1986).
The genome of A. geniculea was sequenced as part of the Darwin Tree of Life Project, a collaborative effort to sequence all named eukaryotic species in the Atlantic Archipelago of Britain and Ireland. Here we present a chromosomally complete genome sequence for A. geniculea, based on one female specimen from Wytham Woods, Oxfordshire, UK.

Genome sequence report
The genome was sequenced from a single female A. geniculea ( Figure 1) collected from Wytham Woods (latitude 51.77, longitude -1.34). A total of 26-fold coverage in Pacific Biosciences single-molecule HiFi long reads and 59-fold coverage in 10X Genomics read clouds were generated. Primary assembly contigs were scaffolded with chromosome conformation Hi-C data. Manual assembly curation corrected 111 missing or mis-joins and removed 40 haplotypic duplications, reducing the assembly length by 1.69% and the scaffold number by 50.81%, and increasing the scaffold N50 by 1.04%.
The final assembly has a total length of 781.6 Mb in 61 sequence scaffolds with a scaffold N50 of 26.4 Mb ( Table 1). Most (99.86%) of the assembly sequence was assigned to 30 chromosomal-level scaffolds, representing 28 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). Some collinearity between Z and W chromosomes can be observed on the Hi-C map. The assembly has a BUSCO v5.3.2 (Manni et al., 2021) completeness of 98.2% (single 97.3%, duplicated 0.9%) using the lepidoptera_odb10 reference set (n = 5,286). While not fully phased, the assembly deposited is of one haplotype. Contigs corresponding to the second haplotype have also been deposited.

Genome annotation report
The A. geniculea GCA_943789525.1 genome assembly was annotated using the Ensembl rapid annotation pipeline (Table 1; Ensembl accession number GCA_943789525.1). The resulting annotation includes 22,347 transcribed mRNAs and 22,132 protein-coding genes.

Sample acquisition and nucleic acid extraction
A female A. geniculea (ilAgrGeni1) was collected in Wytham Woods, Oxfordshire (biological vice-county: Berkshire), UK (latitude 51.77, longitude -1.34) on 9 September 2020 using a light trap. Douglas Boyes (University of Oxford) collected and identified the specimen. The specimen was snap-frozen on dry ice.
DNA was extracted at the Tree of Life laboratory, Wellcome Sanger Institute. The ilAgrGeni1 sample was weighed and dissected on dry ice with tissue set aside for Hi-C sequencing. Whole organism 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.

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 tissue from ilAgrGeni1 using the Arima v2 kit and sequenced on the Illumina NovaSeq 6000 instrument.

Genome assembly
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    et al., 2022)). The assembly was checked for contamination 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., 2022), which performed annotation using MitoFinder (Allio et al., 2020). The genome was analysed and BUSCO scores were generated within the BlobToolKit environment (Challis et al., 2020). Table 3 contains a list of all software tool versions used, where appropriate.

Genome annotation
The BRAKER2 pipeline (Brůna et al., 2021) was used in the default protein mode to generate annotation for the A. geniculea assembly (GCA_943789525.1) in Ensembl Rapid Release.

Ethics/compliance issues
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 . A validation step with macrosynteny against other crambids is recommended to make further proof for the completeness of the assembly, although only 0.14% of the final assembly were not anchored to any chromosome. Therefore, this assembly provides useful genomic resources for further investigations in the evolution of lepidopterans -potentially about the chromosome evolution in the species-rich family Crambidae.

Yes
Competing Interests: No competing interests were disclosed.
Reviewer Expertise: Evolutionary biology, population genomics 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.