The genome sequence of great wood-rush, Luzula sylvatica (Huds) Gaudin

We present a genome assembly from an individual specimen of Luzula sylvatica (great wood-rush; Tracheophyta; Magnoliopsida; Poales; Juncaceae). The genome sequence is 444.5 megabases in span. Most of the assembly is scaffolded into 6 chromosomal pseudomolecules. The mitochondrial and plastid genome assemblies have lengths of 633.36 kilobases and 201.32 kilobases in length, respectively.


Background
The genome of great wood-rush, Luzula sylvatica (Huds.)Gaudin, 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.
Luzula sylvatica is a densely tufted herbaceous perennial (Stace et al., 2019).It is a European temperate species and is widespread throughout Britain and Ireland, although less common in central and eastern England and central Ireland (Stroh et al., 2023).Its range in Britain and Ireland is stable, although it has declined in central and eastern England since the 1960s (Stroh et al., 2023).
It is shade tolerant and typically found in woodlands, moorlands, stream sides, and on montane ledges.It has a broad altitudinal range, from sea-level to 1040 m (Stroh et al., 2023).The flowers are hermaphrodite and observations on other Luzula species show that although the predominant mode of pollination is wind pollination, insect pollination may also occur (Huang et al., 2013).No hybrids of Luzula are recorded from Britain and Ireland (Stace et al., 2015).
Within Britain and Ireland the species is diploid (2n = 12), with chromosome counts made on native material from three different populations in England, Ireland and Scotland (Dempsey et al., 1994;Gornall & Bailey, 1993).In this paper we present a high-quality reference genome as a foundation resource for future studies.

Genome sequence report
The genome was sequenced from Luzula sylvatica (Figure 1) collected from Royal Botanic Garden Edinburgh (Inverleith), Scotland, UK (55.97,.Using flow cytometry, the genome size (1C-value) was estimated to be 0.58 pg, equivalent to 580 Mb.A total of 33-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 93 missing joins or mis-joins and removed 7 haplotypic duplications, reducing the assembly length by 1.13% and the scaffold number by 50.65%, and increasing the scaffold N50 by 1.12%.
The final assembly has a total length of 444.5 Mb in 36 sequence scaffolds with a scaffold N50 of 74.5 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 (96.69%) of the assembly sequence was assigned to 6 chromosomal-level scaffolds.Chromosome-scale scaffolds confirmed by the Hi-C data are named in order of size (Figure 5; Table 2).Heterozygous inversions were observed on chromosome 5, in the region of 7.3 Mb to 22.9 Mb.While not fully phased, the assembly deposited is of one haplotype.Contigs corresponding to the second haplotype have also been deposited.The mitochondrial and plastid genomes were also assembled and can be found as contigs within the multifasta file of the genome submission.

Sample acquisition, genome size estimation and nucleic acid extraction
Luzula sylvatica (specimen ID EDTOL00071, ToLID lpLuz-Sylv1) was collected from Royal Botanic Garden Edinburgh (Inverleith), Scotland, UK (latitude 55.97, longitude -3.21) on  2020-08-12.The plant was originally collected in 1993 from the Scottish highlands (Glenmore, Coire na Ciste), at an altitude of 600 m.The specimen was collected and formally identified by Zoe Goodwin and David Bell (Royal Botanic Garden Edinburgh).Leaves were cut into segments using scissors and snap-frozen in liquid nitrogen.The herbarium specimen of the sequenced plant is kept at the Royal Botanic Garden Edinburgh (E) https://data.rbge.org.uk/herb/E01358005.
The genome size was estimated by flow cytometry using the fluorochrome propidium iodide and following the 'one-step' method as outlined in Pellicer et al. (2021).For this species, the General Purpose Buffer (GPB) supplemented with 3% PVP and 0.08% (v/v) beta-mercaptoethanol was used for isolation of nuclei (Loureiro et al., 2007), and the internal calibration standard was Solanum lycopersicum 'Stupiké polní rané' with an assumed 1C-value of 968 Mb (Doležel et al., 2007).et al., 2023).HMW DNA was extracted using the Automated Plant MagAttract v2 protocol (Todorovic et al., 2023a).HMW DNA was sheared into an average fragment size of 12-20 kb in a Megaruptor 3 system with speed setting 30 (Todorovic et al., 2023b).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.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 leaf tissue of lpLuzSylv1 using the Arima2 kit and sequenced on the Illumina NovaSeq 6000 instrument.
A Hi-C map for the final assembly was produced using bwa-mem2 (Vasimuddin et al., 2019)  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   The paper reads well, and present a good contribution to reference genome development for the grass spp.
Having said this, what is the main use of this grass?Please add a sentence or two on the importance of the crop in the introduction section.
In the result section, the genome assembly was presented.No genome annotation and gene family analyses were done.If possible, for better completeness of the information, please add annotation analysis.

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: genomic/genetic analysis, plant biotechnology, data analysis, genebank, quantitative genetics, field evaluation, phenotyping 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 1 .
Figure 1.Photograph of the herbarium voucher of the Luzula sylvatica (lpLuzSylv1) specimen used for genome sequencing.

Figure 2 .
Figure 2. Genome assembly of Luzula sylvatica, lpLuzSylv1.1: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 445,370,405 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 (77,244,432 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (74,526,441 and 60,551,003 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 poales_ odb10 set is shown in the top right.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/CAMPEJ01/dataset/CAMPEJ01/snail.

Figure 3 .
Figure 3. Genome assembly of Luzula sylvatica, lpLuzSylv1.1: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/CAMPEJ01/dataset/CAMPEJ01/blob.

Figure 4 .
Figure 4. Genome assembly of Luzula sylvatica, lpLuzSylv1.1: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/CAMPEJ01/dataset/CAMPEJ01/cumulative.

Figure 5 .
Figure 5. Genome assembly of Luzula sylvatica, lpLuzSylv1.1:Hi-C contact map of the lpLuzSylv1.1 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=OgFWtEaJQnGD2mhY8BD_Cw.

Table 3 . Software tools: versions and sources. Software tool Version Yanis Bouchenak-Khelladi University
of Bourgogne Franche-Comté, INRAE, Dijon, France, Dijon, France This paper is well written, sufficiently detailed and the genome produced is of very good quality.All datasets are easily accessible and in useable format.I found one typo: "...sample preparation;..." use colon instead of semicolon in the Methods_Sample acquisition section.Nothing else to report, it is a nice data paper.

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.
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.Feed and Forage Development Program, International Livestock Research Institute Ethiopia, Addis Ababa, Addis Ababa, Ethiopia https://doi.org/10.21956/wellcomeopenres.23232.r89823© 2024 Negawo A.