The genome sequence of the Fruity Milkcap, Lactarius evosmus (Kühner & Romagn., 1954)

We present a genome assembly from an individual Lactarius evosmus (Fruity Milkcap; Basidiomycota; Agaricomycetes; Russulales; Russulaceae). The genome sequence is 57.2 megabases in span. Most of the assembly is scaffolded into 11 chromosomal pseudomolecules. The mitochondrial genome has also been assembled and is 61.51 kilobases in length.


Background
Lactarius evosmus (Fruity Milkcap) is a member of a large genus of mycorrhizal mushrooms well represented in the UK and widespread in Europe.The order Russulales comprises Lactarius and Russula -both gilled genera -together with some bracket, resupinate, and toothed fungi, and even some truffles.All members of this order of fungi have spores with strongly amyloid ornamentation (turning blue-black in Melzer's reagent).The genus Lactarius has one unique feature: damage to the gills promptly produces the tell-tale 'milk' (latex), hence its common English name, Milkcap.The latex -white or occasionally colourless when first exposed to air -often changes colour to yellow, pink, greenish-grey, or even violet-purple, depending on the species.Ecologically, some Lactarius species associate with deciduous trees, others with conifers, some are host specific, and still others are generalists (Kibby, 2020).
Lactarius evosmus is ectomycorrhizal with Quercus, Populus, and Salix species, favouring calcareous soils, although it is not one of the UK's more common Milkcap species.Since the late 1990s, we have become aware of it fruiting prolifically in association with Helianthemum nummularium (Rock Rose) in open chalk downland with no trees presentone of very few members of this genus to do this -though there are growing numbers of other mycorrhizal genera well represented in such habitats.The specimen used for genome sequencing was collected from Watlington Hill on the Chiltern escarpment in Oxfordshire in association with Helianthemum.The species can regularly be seen here in extraordinary numbers during the autumn months together with a range of other mushroom species.
Lactarius evosmus is a medium to large Milkcap (Figure 1).The cap is 6-10(15) cm across, tightly inrolled at first, then becoming less so as it expands with a sunken centre, surface ± smooth, sticky in damp conditions, cream to yellowish claybuff and developing distinct radial darker zones more ochrebrown as it matures.The gills are fairly crowded, adnate to slightly decurrent or with decurrent tooth; at first cream, later pinkish buff.The latex is abundant, remaining white as it dries, with very acrid taste.Stem to 6 × 3 cm in dimension, dry, firm and smooth, pale cream, becoming more ochre with age.The smell is significantly fruity, reminiscent of apples and similar to that in Russula fellea -hence its common species name.The sporeprint is pinkish-buff.The basidia are 4-spored.The spores average 8 × 6.5 µm, ellipsoid, and are ornamented with low warts and ridges, which are amyloid.Cystidia are present on the gill edge but are not considered significant for species identification (Noordeloos et al., 2018).
The species is very similar in appearance to both L. acerrimus and L. zonarius.The first, however, has 2-spored basidia; the second has a notably pubescent margin in early stages and a stem with small 'pock-marks'.All three species can occur under Quercus, but only L. evosmus has the distinct apple smell.To date neither L. acerrimus nor L. zonarius occur with Helianthemum.
The genome of Lactarius evosmus 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 complete chromosomal genome sequence for a Lactarius evosmus specimen from Watlington Hill, found in association with Helianthemum nummularium.

Genome sequence report
The genome was sequenced from a specimen of Lactarius evosmus collected from Watlington Hill, Oxfordshire (51.64, -0.99).A total of 326-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 three missing joins or misjoins and removed one haplotypic duplication, reducing the assembly length by 22.58% and the scaffold number by 98.18%, and increasing the scaffold N50 by 10.85%.
The final assembly has a total length of 57.2 Mb in 12 sequence scaffolds with a scaffold N50 of 5.4 Mb (Table 1).Most (99.89%) of the assembly sequence was assigned to 11 chromosomal-level scaffolds.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.
Metadata for specimens, spectral estimates, sequencing runs, contaminants and pre-curation assembly statistics can be found at https://links.tol.sanger.ac.uk/species/326039.

Sample acquisition and nucleic acid extraction
A Lactarius evosmus (specimen ID KDTOL00275, ToLID gfLacEvos1) was collected from Watlington Hill, Oxfordshire, UK (latitude 51.64, longitude -0.99) on 2021-11-02.The specimen was handpicked from an area with Helianthemum sp. by Penny Cullington and transported to the Royal Botanic Gardens, Kew for snap freezing.The specimen was identified by Penny Cullington (British Mycological Society), confirmed by Brian Douglas (Royal Botanic Gardens Kew) and processed and snap-frozen in liquid nitrogen by Kieran Woof.
DNA was extracted at the Tree of Life laboratory, Wellcome Sanger Institute (WSI).The gfLacEvos1 sample was weighed RNA was extracted from cap tissue of gfLacEvos1 in the Tree of Life Laboratory at the WSI using TRIzol, according to the manufacturer's instructions.RNA was then eluted in 50 µl RNAse-free water and its concentration assessed using a Nanodrop spectrophotometer and Qubit Fluorometer using the Qubit RNA Broad-Range (BR) Assay kit.Analysis of the integrity of the RNA was done using Agilent RNA 6000 Pico Kit and Eukaryotic Total RNA assay.

Sequencing
Pacific Biosciences HiFi circular consensus DNA sequencing libraries were constructed according to the manufacturers' instructions.Poly(A) RNA-Seq libraries were constructed using the NEB Ultra II RNA Library Prep kit.DNA and RNA sequencing was performed by the Scientific Operations core at the WSI on Pacific Biosciences SEQUEL II
A Hi-C map for the final assembly was produced using bwa-mem2 (Vasimuddin et al., 2019) et al., 2015) were calculated.
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 (HiFi) and Illumina NovaSeq 6000 (RNA-Seq) instruments.Hi-C data were also generated from cap tissue of gfLacEvos1 using the Arima2 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).The assembly was then scaffolded with Hi-C data (Rao et al., 2014) using YaHS 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 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

Bhagya C. Thimmappa
Université de Montréal, Montréal, Canada In the work titled "The Genome Sequence of the Fruity Milkcap, Lactarius evosmus," the authors report nuclear and mitochondrial assembly for Lactarius evosmus, which was initially collected from Watlington Hill.

Comments:
What  Major comments: 1) The rationale for creating the datasets appears to be "The genome of Lactarius evosmus 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." Please provide some details regarding what are the actual questions of interest, how this genome fits into the broader scheme of things, such as other genomes being sequenced etc.
2) Some context for the genome assembly metrics and how it compared to either other species with similar genomes or to the best genome assembly possible would be important.
Unfortunately, no effort is made to evaluate the "validity" of the genome assembly.Have you considered using some post-assembly validation tools like Reaper?
3) Comparing the assembly structure with the genome of other closely related species should also provide some idea of how well the assembly has performed 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

Figure 2 .
Figure 2. Genome assembly of Lactarius evosmus, gfLacEvos1.1: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 57,293,171 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 (6,657,085 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (5,448,807 and 4,650,992 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 agaricomycetes_odb10 set is shown in the top right.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/gfLacEvos1.1/dataset/ CATOBD01/snail.

Figure 3 .
Figure 3. Genome assembly of Lactarius evosmus, gfLacEvos1.1: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/gfLacEvos1.1/dataset/CATOBD01/blob.

Figure 4 .
Figure 4. Genome assembly of Lactarius evosmus, gfLacEvos1.1: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/gfLacEvos1.1/dataset/CATOBD01/ cumulative.

Figure 5 .
Figure 5. Genome assembly of Lactarius evosmus, gfLacEvos1.1:Hi-C contact map of the gfLacEvos1.1 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=a7wK8basTWa4jP_ndUAizg.

©
2024 Vijay N.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.Nagarjun Vijay Indian Institute of Science Education and Research (IISER), Bhopal, MP, India Summary of the article: Cullington et al., generate a high quality genome assembly of the Fruity Milkcap, Lactarius evosmus.The genome is assembled using 326x coverage PacBio along with Hi-C data.Standard genome assembly metrics such as the BlobToolKit Snailplot, GC-coverage plot, cumulative sequence plot, Hi-C contact map are provided.

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 those missing Buscos?What does it tell us about Lactarius evosmus?○Is there a phylogenetic tree showing how Lactarius evosmus is placed with other closely related species?In other words, what are other closely related species?○ Reported mitochondrial genome assembly could be a valuable resource for building better phylogenetic trees (provided mitochondrial genome assembly is available for other species).Reviewer Expertise: Fungal genomics and transcriptomicsI 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.
https://doi.org/10.21956/wellcomeopenres.22046.r83759