The genome sequence of the oyster mushroom, Pleurotus ostreatus ((Jacq.) P. Kummer, 1871)

We present a genome assembly from a Pleurotus ostreatus specimen (the oyster mushroom; Basidiomycota; Agaricomycetes; Agaricales; Pleurotaceae). The genome sequence is 40.6 megabases in span. Most of the assembly is scaffolded into 12 chromosomal pseudomolecules. Two mitochondrial genomes have been assembled, which are 73.1 and 9.3 kilobases in length.


Background
Pleurotus ostreatus (oyster mushroom) is a lamellate fungus that produces thin, fan-shaped, laterally-attached, fleshy sporocarps, with a dark grey, grey brown, bluish grey to yellow brown pileus, and an eccentric stem with strongly decurrent white lamellae.They grow in dense, imbricate clusters with each cap ranging in size from 50-150(-350) mm.
In culture, the hyphae are hyaline, clamped and branched, with considerable aerial growth, and can produce subglobose to broadly ellipsoid thick-walled chlamydospores in aging cultures.In low nitrogen conditions, the hyphae can produce secretory cells that excrete powerful toxins that paralyse nematodes and cause shrinkage of their heads, before hyphal colonisation and digestion (Soares et al., 2018).This species grows rapidly and aggressively in culture and is capable of producing sporocarps in short periods of time on a wide range of cellulose and lignin-based substrates.This, combined with desirable culinary characteristics and good keeping qualities, has led to P. ostreatus being one of the three most extensively cultivated fungi in the world (Li et al., 2020;Royse et al., 2017).
Pleurotus ostreatus has a global distribution that includes all continents except Antarctica (Li et al., 2020).Throughout Europe it is known to cause a white-rot in the wood of a broad range of angiosperm trees, particularly Fagus and Quercus, either on standing trunks or larger fallen branches.In the UK, P. ostreatus is widespread and common wherever host trees are present.
Oyster mushrooms have been shown to produce a diverse and valuable range of enzymes with biotechnological, therapeutic, and bioremediation uses (Corrêa et al., 2016;Knop et al., 2015), and further discoveries, alongside improved taxonomic understanding of the cryptically diverse group will be aided by the production of this genome.

Genome sequence report
The genome was sequenced from a Pleurotus ostreatus specimen (Figure 1) grown in a pure culture obtained from a single sporocarp collected at Eastville Park, Bristol, UK (latitude 51.48, longitude -2.56).A total of 57-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 four missing joins or mis-joins and removed one haplotypic duplication, increasing the scaffold count by three.
The final assembly has a total length of 40.6 Mb in 14 sequence scaffolds with a scaffold N50 of 4.3 Mb (Table 1).Most (99.79%) of the assembly sequence was assigned to 12 chromosomal-level scaffolds.Chromosome-scale scaffolds confirmed by the Hi-C data are named in order of size (Figure 2-Figure 5; Table 2).There is a large heterozygous inversion on chromosome 5 from 569.3 kb to 2.176 Mb (Figure 5).While not fully phased, the assembly deposited is of one haplotype.Contigs corresponding to the second haplotype have also been deposited.The mitochondrial genomes were also assembled and can be found as a contig within the multifasta file of the genome submission.
Metadata for specimens, spectral estimates, sequencing runs, contaminants and pre-curation assembly statistics can be found at https://links.tol.sanger.ac.uk/species/5322.

Sample acquisition and nucleic acid extraction
A Pleurotus ostreatus specimen (gfPleOstr1) was collected at Eastville Park, Bristol, UK (latitude 51.48, longitude -2.56) by Richard Wright (RBGK, Cardiff University).The specimen was grown in pure culture obtained from a single sporocarp by Richard Wright.Culture handling and initial barcoding to confirm identity was carried out by Kieran Woof (RBGK) and samples taken from it were preserved on dry ice.
DNA was extracted at the Tree of Life laboratory, Wellcome Sanger Institute (WSI).The gfPleOstr1 sample was weighed and dissected on dry ice with tissue set aside for Hi-C sequencing.Tissue was cryogenically disrupted to a fine powder using a Covaris cryoPREP Automated Dry Pulveriser, receiving multiple impacts.High molecular weight (HMW) DNA was extracted using the Qiagen Plant MagAttract 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 a Pacific Biosciences SEQUEL II (HiFi) instrument.Hi-C data were also generated from tissue of gfPleOstr1 using the Arimav2 kit and sequenced on the Illumina NovaSeq 6000 instrument.and to ensure the general quality of the sequence.The mitochondrial genome was assembled using MitoHiFi.The mitochondrial linear plasmid MT2 was assembled using MBG (Rautiainen & Marschall, 2021).
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   Further, the Wellcome Sanger Institute employs a process whereby due diligence is carried out proportionate to the nature of the materials themselves, and the circumstances under which they have been/are to be collected and provided for use.The purpose of this is to address and mitigate any potential legal and/or ethical implications of receipt and use of the materials as part of the research project, and to ensure that in doing so we align with best practice wherever possible.The overarching areas of consideration are: • Ethical review of provenance and sourcing of the material

Erich M. Schwarz
Cornell University, Ithaca, New York, USA This description of a Pleurotus ostreatus genome assembly is concise, complete, and credible.The genome has been properly archived in GenBank, the methods described are appropriate, and both the raw data and the software tools are rigorously described with URLs for data or code files.
I observe only one error (which a previous reviewer pointed out a year ago!).In the Methods, there is a second copy of the sentence "The mitochondrial genome was assembled using MitoHiFi.",immediately before the sentence "The mitochondrial linear plasmid MT2 was assembled using MBG...".This second sentence about using MitoHiFi is redundant and should be deleted.
I also note that in their Data Availability section, the authors promise, "The genome will be annotated using available RNA-Seq data and presented through the Ensembl pipeline at the European Bioinformatics Institute."There may exist RNA-seq data for Pleurotus ostreatus, but the authors do not provide a citation or web link for them, and it is not clear if this annotation will ever happen (I did not find any such annotation in Ensembl on 21 May 2025).It would be good if such RNA-seq data really existed and was used to provide protein-coding gene predictions for this highquality fungal genome.

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
Are the datasets clearly presented in a useable and accessible format?Yes Competing Interests: No competing interests were disclosed.
Reviewer Expertise: Genomics, epigenetics, fungal 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.

Figure 1 .
Figure 1. A. Pleurotus ostreatus sporocarps, growing on the trunk of Aesculus hippocastanum.B. The underside of the same sporocarps showing lamellae.

Figure 2 .
Figure 2. Genome assembly of Pleurotus ostreatus, gfPleOstr1.1: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 40,636,420 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 (5,861,466 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (4,316,464 and 2,120,766 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 agaricales_odb10 set is shown in the top right.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/gfPleOstr1.1/dataset/ CAMQQE01/snail.

Figure 3 .
Figure 3. Genome assembly of Pleurotus ostreatus, gfPleOstr1.1: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/gfPleOstr1.1/dataset/CAMQQE01/blob.

Figure 4 .
Figure 4. Genome assembly of Pleurotus ostreatus, gfPleOstr1.1: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/gfPleOstr1.1/dataset/CAMQQE01/ cumulative.

Figure 5 .
Figure 5. Genome assembly of Pleurotus ostreatus, gfPleOstr1.1:Hi-C contact map of the gfPleOstr1.1 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=RUr4Tdg_SNqA3C4JO5x1cA.