The genome sequence of the Green-brindled Crescent, Allophyes oxyacanthae (Linnaeus, 1758)

We present a genome assembly from an individual male Allophyes oxyacanthae (the Green-brindled Crescent; Arthropoda; Insecta; Lepidoptera; Noctuidae). The genome sequence is 458 megabases in span. The whole assembly is scaffolded into 31 chromosomal pseudomolecules, including the assembled Z sex chromosome. The mitochondrial genome has also been assembled and is 15.3 kilobases in length. Gene annotation of this assembly on Ensembl has identified 17,301 protein coding genes.


Background
The Green-brindled Crescent Allophyes oxyacanthae is a moth in the family Noctuidae with an autumn flight period in the UK.The typical form of the moth is unmistakable, with a dense scattering of shimmering metallic green scales on the forewings, contrasting against a deep brown ground colour, pale marginal band and pale orbicular and reniform stigmata (oval and kidney marks).A distinct colour variant is also encountered in the UK, denoted form capucina, with uniform brown colouration and fewer green scales.Breeding experiments suggest that the difference between the two forms is controlled by a single genetic locus, with the f.capucina allele dominant to the wild type allele (Steward, 1977).It has been suggested that f. capucina moths may have had a selective advantage in areas of industrial pollution, although the data are unclear on this issue (Ford, 1967).The genetic locus and molecular basis of the polymorphism have not been identified.
A. oxyacanthae has been recorded across most of UK, including the north of Scotland, through Scandinavia and across mainland Europe although there are few records from Italy and southern Spain (GBIF Secretariat, 2022).The species is attracted to light and can be found in woodlands and gardens where the larval food plants of hawthorn, blackthorn, rowan and fruit trees are found (Robinson et al., 2010).
A genome sequence for A. oxyacanthae will facilitate study of the genetic basis of colour polymorphism and the molecular adaptations underpinning polyphagy, and contribute to a growing data set of resources for understanding lepidopteran biology.

Genome sequence report
The genome was sequenced from one male Allophyes oxyacanthae specimen (Figure 1) collected in Wytham Woods (latitude 51.77, longitude -1.34).A total of 71-fold coverage in Pacific Biosciences single-molecule HiFi long reads weas generated.Primary assembly contigs were scaffolded with chromosome conformation Hi-C data.Manual assembly curation corrected three missing or mis-joins, reducing the scaffold number by 8.82%.
The final assembly has a total length of 458.5 Mb in 31 sequence scaffolds with a scaffold N50 of 16.7 Mb (Table 1).The whole assembly sequence was assigned to 31 chromosomal-level scaffolds, representing 30 autosomes and the Z sex chromosome.Chromosome-scale scaffolds confirmed by the Hi-C data have been 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 99.2% (single 98.8%, duplicated 0.4%) using the lepidoptera_odb10 reference set (n = 5286).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. oxyacanthae genome assembly (GCA_932294325.1)was annotated using the Ensembl rapid annotation pipeline (Table 1).The resulting annotation includes 17,485 transcribed mRNAs from 17,301 protein-coding genes.

Sample acquisition and nucleic acid extraction
An individual male A. oxyacanthae specimen (ilAllOxya1) was collected in Wytham Woods, Oxfordshire (biological vice-county: Berkshire), UK (latitude 51.77, longitude -1.34) on 8 October 2020 using a light trap.The specimens were collected and identified by Douglas Boyes (University of Oxford) and snap-frozen on dry ice.This specimen was used for DNA and Hi-C sequencing.
DNA was extracted at the Tree of Life laboratory, Wellcome Sanger Institute (WSI).The ilAllOxya1 sample was weighed and dissected on dry ice with head 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.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 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) instrument.Hi-C data were also generated from head  (Uliano-Silva et al., 2022), which performed annotation using MitoFinder (Allio et al., 2020).The genome was analysed and BUSCO scores 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 Allophyes oxyacanthae assembly (GCA_934047225.1) in Ensembl Rapid Release.

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

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: Genomics; Genetics and Evolution ; Insects 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.

Amali Thrimawithana
Plant and Food Research Institute of New Zealand Ltd, Auckland, New Zealand This article summarise the assembly and annotation of the Green-brindled Crescent, which is great to see.As it helps articulate the how the species colour polymorphism make work at a molecular level as well as add to the lepidopteran genome databases.
One thing I was keen to know was why is the colour variant is of importance?Though the background highlighted the need for it given currently low knowledge on the aspect, I failed to understand how the knowledge on the colour polymorphism might help?Would it be related to management of the species?
Within the methods, I was wondering what sort of evidence was used to run the annotation through BRAKER pipeline?Did you have RNASeq data or protein data from similar species?This would be a useful information to add.If the data like RNASeq was generated by the project for this -it will need to be added to method section too.
One other minor addition I thought might be useful is to mention the database used by Busco for completion checking of the genome -(I can see it in Figure 2, but thought would be good to have in the method section too).
All in all, great to see more high quality lepidopteran genomes coming out -building great resource for lepidopteran studies.

Is the rationale for creating the dataset(s) clearly described? Partly
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 Temperate Tree Fruit and Vegetable Research Unit, USDA-ARS, Wapato, Washington, USA The authors present a report on the genome sequence of the Green-brindled Crescent moth, Allophyes oxyacanthae.The manuscript is well written and straightforward, adhering to standards for this type of report, including information on predicted gene annotations.
This manuscript was previously examined by another reviewer, and I am inclined to agree with all comments of the previous reviewer, and would especially emphasize that I agree with the request for more detailed information on parameters/conditions for all of the bioinformatics software that were used.
Aside from that, I have no further comments, as the manuscript is otherwise very suitable for indexing.

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? No
Are the datasets clearly presented in a useable and accessible format?Yes Competing Interests: No competing interests were disclosed.
Reviewer Expertise: Insect genetics, genomics and molecular biology.
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, however I have significant reservations, as outlined above.
Reviewer Report 03 August 2023 https://doi.org/10.21956/wellcomeopenres.20993.r63371 (Linnaeus, 1758)" is well written and all the information are clearly presented and the aim of the paper is to provide useful information to further studies genetic basis of color variations in the study species.
Is there any available information about genome size of the study species or sister species based on direct method such as FACS? 1.
Can the authors provide detailed information how all programs were executed?2.
Is there any accession number for the proteome?4.

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? No
Are the datasets clearly presented in a useable and accessible format?Yes Competing Interests: No competing interests were disclosed.
Reviewer Expertise: Genomics, transcriptomics and proteomics 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, however I have significant reservations, as outlined above.

Figure 2 .
Figure 2. Genome assembly of Allophyes oxyacanthae, ilAllOxya1.1: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 458,479,537 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 (20,278,968 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (16,683,655 and 10,717,387 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/ilAllOxya1_1/dataset/ilAllOxya1_1/snail.

Figure 3 .
Figure 3. Genome assembly of Allophyes oxyacanthae, ilAllOxya1.1: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/ilAllOxya1_1/dataset/ilAllOxya1_1/blob.

Figure 4 .
Figure 4. Genome assembly of Allophyes oxyacanthae, ilAllOxya1.1:cumulative sequence.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/ilAllOxya1_1/dataset/ilAllOxya1_1/cumulative.

Figure 5 .
Figure 5. Genome assembly of Allophyes oxyacanthae, ilAllOxya1.1:Hi-C contact map.Hi-C contact map of the ilAllOxya1.1 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=OzgdewECSHeyOqN4f7ptEQ.

Reviewer Report 14
November 2023 https://doi.org/10.21956/wellcomeopenres.20993.r63373© 2023 Thrimawithana A. 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. Software tool Version Source
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.