Genome assembly of the edible jelly fungus Dacryopinax spathularia (Dacrymycetaceae)

The edible jelly fungus Dacryopinax spathularia (Dacrymycetaceae) is wood-decaying and can be commonly found worldwide. It has found application in food additives, given its ability to synthesize long-chain glycolipids, among other uses. In this study, we present the genome assembly of D. spathularia using a combination of PacBio HiFi reads and Omni-C data. The genome size is 29.2 Mb. It has high sequence contiguity and completeness, with a scaffold N50 of 1.925 Mb and a 92.0% BUSCO score. A total of 11,510 protein-coding genes and 474.7 kb repeats (accounting for 1.62% of the genome) were predicted. The D. spathularia genome assembly generated in this study provides a valuable resource for understanding their ecology, such as their wood-decaying capability, their evolutionary relationships with other fungi, and their unique biology and applications in the food industry.


INTRODUCTION
Dacryopinax spathularia (Dacrymycetaceae, NCBI:txid139277) (Figure 1A) is a brown-rot fungus commonly found on rotting coniferous and broadleaf wood worldwide.This fungus can be easily distinguished by the spathulate shape of its gelatinous fruiting body [1,2].Owing to its production of carotenoid pigments as a protection against UV damage, its external appearance is generally orange to yellow [3].In addition to its ecological role in nutrient recycling, this species is also edible and commonly known as the "sweet osmanthus ear" mushroom in China [4].Given its ability to synthesise long-chain glycolipids under fermentation, this species has also been cultivated in the food industry to produce natural preservatives for soft drinks [5].

CONTEXT
Edible jelly fungus D. spathularia (Dacrymycetaceae), which was first described as Merulius spathularius, is a macrofungus basidiomycete and can be commonly found on rotting coniferous and broadleaf wood in tropics and subtropics.Its wood-decaying ability facilitates nutrient recycling in forest ecosystems [6].This species is edible and frequently cultivated in industry to produce food additives such as natural preservatives for soft drinks [4,7].In addition, the isolated fungal extract can also display anti-bacterial properties [8].D. spathularia can be naturally found in Asia, Africa, America, Australia and other parts of the Pacific region.To date, the genomic data of the genus Dacryopinax is limited to Dacryopinax primogenitus, which is used for studying the origin of genes involved in lignin decomposition among different wood-decaying fungi lineages [9].However, the genomic data of D. spathularia is not available.
In Hong Kong, D. spathularia can be commonly found [10] and has been selected as one of the species to be sequenced by the Hong Kong Biodiversity Genomics Consortium (also known as EarthBioGenome Project Hong Kong) formed by investigators from eight publicly funded universities.Here, we present the genome assembly of D. spathularia, which was assembled from PacBio long reads and Omni-C sequencing data.The D. spathularia genome will help better understand this fungus' ecology, the genetic basis of its wood-decaying ability, the phylogenetic relationships in its family and the biosynthesis of the long-chain glycolipids that are used as natural preservatives in the food industry.

Sample collection and culture of fungal isolates
The fruiting bodies of D. spathularia were collected in Luk Keng, Hong Kong, on 20 June 2022 (Figure 1A).The fungal isolate was transferred from the edge of fruit bodies to potato dextrose agar (BD Difco™) plates using a pair of sterilized forceps.The remaining collected fruit bodies were snap-frozen with liquid nitrogen and stored in a −80 °C freezer.Fungal hyphae from 2-week-old colonies were transferred to new plates for purification for at least three rounds.The identity of the isolate, termed "F14", was assigned by DNA barcoding using the sequence of the Translation elongation factor 1 alpha (TEF-1α) gene using the primer pairs EF1-1018F and EF1-1620R [11] (Figure 1B).

High molecular weight DNA extraction
Approximately 1.5 g of mycelia of D. spathularia isolate was collected from the upper layer of the agar culture and ground in a mortar with liquid nitrogen.High molecular weight (HMW) genomic DNA was isolated with cetyltrimethylammonium bromide (CTAB) treatment, followed by the NucleoBond HMW DNA kit (Macherey Nagel Item No. 740160.20).Briefly, the ground tissue was transferred to 5 mL CTAB buffer [12] with an addition of 1% Polyvinylpyrrolidone for 1 hour digestion at 55 °C.After RNAse A treatment, by adding 100 μL RNAse A and incubating for 10 min at room temperature, 1.6 mL 3M potassium acetate was added to the lysate.The lysate was then aliquoted into six 2 mL tubes (each containing ∼1.1 mL lysate).Next, 800 μL chloroform:IAA (24:1) was added to each tube and gently mixed by inverting the tubes for ∼10 s, followed by centrifugation at >10,000 × g for 5 min.The supernatant (∼900 μL from each tube) was transferred to a new tube and 800 μL chloroform:IAA (24:1) was added for another round of wash with the same procedure of mixing and centrifugation.Subsequently, the supernatant (∼800 μL from each tube, ∼4.8 mL total) was mixed with ∼1.2 mL H1 buffer from the NucleoBond HMW DNA kit for a final volume of 6 mL and processed according to the manufacturer's protocol.The DNA sample was eluted in 80 μL elution buffer (PacBio Ref. No. 101-633-500) and its quantity and quality were assessed with NanoDrop™ One/OneC Microvolume UV-Vis Spectrophotometer, Qubit ® Fluorometer, and overnight pulse-field gel electrophoresis (Figure 1C).

PacBio library preparation and sequencing
DNA shearing was first performed from 5 μg HMW DNA in 120 μL elution buffer using a g-tube (Covaris Part No. 520079) with six centrifugation steps at 1,990 × g for 2 min.The sheared DNA sample was purified using SMRTbell ® cleanup beads (PacBio Ref. No. 102158-300).Also, 2 μL of the DNA sample was used for quality check through overnight pulse-field gel electrophoresis and Qubit ® Fluorometer quantification.An SMRTbell library      1.

RNA extraction and transcriptome sequencing
Approximately 1 g of mycelia of D. spathularia isolate was ground in a mortar with liquid nitrogen.Total RNA was isolated from the ground tissue using the mirVana miRNA Isolation Kit (Ambion), following the manufacturer's instructions.The RNA sample underwent quality control with NanoDrop™ One/OneC Microvolume UV-Vis Spectrophotometer and 1% agarose gel electrophoresis.Finally, the qualified sample was sent to Novogene Co. Ltd (Hong Kong, China) for 150 bp paired-end sequencing.Details of the resulting sequencing data are listed in Table 1.

Genome assembly and gene model prediction
A de novo genome assembly was conducted with Hifiasm [13], which was screened with BlobTools (v1.1.1)[14] by searching against the NT database using blastn (RRID:SCR_004870) to identify and remove any possible contaminations.Haplotypic duplications were discarded using purge_dups (RRID:SCR_021173) according to the depth of the HiFi reads [15].The Omni-C data were used to scaffold the assembly using YaHS [16].

Repeat annotation
Transposable element (TE) annotation was performed by following the Earl Grey TE annotation workflow pipeline (version 1.2) [21].

Genome assembly
A total of 9.34 Gb HiFi reads was generated from PacBio sequencing (Table 1).After scaffolding with 3.46 Gb Omni-C data, the D. spathularia genome assembly has a size of 29.2 Mb, scaffold N50 of 1.925 Mb and 92.0%BUSCO (RRID:SCR_015008) score [22] (Figure 2 and Table 2), and 19 out of 24 scaffolds are >100 kb in length and validated by inspection of the Omni-C contact maps (Figure 1C and Table 2).The genome size is similar to D. primogenitus (29.5 Mb) [9] (Figure 1B) and GenomeScope (RRID:SCR_017014) estimated heterozygosity of 5.09% (Figure 1D; Table 3).Gene model prediction generated a total of 11,510 protein-coding genes with an average protein length of ∼451 amino acids and a BUSCO score of 91.9%.

CONCLUSION AND FUTURE PERSPECTIVE
The study presents the genome assembly of D. spathularia, a useful resource for further phylogenomic studies in the family Dacrymycetaceae and investigations on the biosynthesis of glycolipids with potential applications in the food industry.

DATA VALIDATION AND QUALITY CONTROL
The identity of the fungal isolate of D. spathularia was validated with the DNA barcoding of the TEF-1α gene, which was compared with sequences from phylogenetic studies of Dacrymycetaceae [23] and their sister family Cerinomycetaceae [24], the D. primogenitus genome (Accession: NW_024467206.1B).
For HMW DNA extraction and Pacbio library preparation, the samples were subject to quality control with NanoDrop™ One/OneC Microvolume UV-Vis Spectrophotometer, Qubit ® Fluorometer, and overnight pulse-field gel electrophoresis (Figure 1C).The quality of the Omni-C library was inspected with Qubit ® Fluorometer and TapeStation D5000 HS ScreenTape.
Finally, GenomeScope2 [28] was used to estimate the genome size and heterozygosity of the assembly.

Figure 1 .
Figure 1.Sample information of D. spathularia.(A) Picture of the D. spathularia we collected in the field; (B) Phylogenetic analysis of the TEF-1α gene region in the D. spathularia fungal isolate "F14" of this study.Sequences from related phylogenetic studies and NCBI accessions were incorporated, including Zaroma & Ekman [23], Savchenko et al. [24], the D. primogenitus genome (NW_024467206.1:736197-736766) and the NCBI BLAST result of a D. spathularia isolate (Accession: AY881020.1),which are highlighted in light blue, pink, green, and red, respectively.The fungal isolate "F14" is highlighted in orange.The bootstrap percentage values are shown at the nodes.The sequence alignment and tree file can be retrieved from [29]; (C) Information on the quality control of the extracted high molecular weight DNA sample.The left panel shows the photograph of the overnight pulse-field gel electrophoresis of the extracted DNA sample together with a 15 kb DNA ladder (15 kb DL) and Lambda-Hind III Digest marker (Lambda DM).The right panel summarizes the information on Qubit ® Fluorometer and NanoDrop™ One/OneC Microvolume UV-Vis Spectrophotometer.Abbreviation: conc., concentration.

Figure 2 .
Figure 2. (A) Genome statistics; (B) Omni-C contact map of the assembly.The chromatin contact intensities are indicated in red along the matrix of genomic regions, and the boundary of scaffolds are defined by black lines; (C) GenomeScope report summary; (D) Repeat landscape plot (left) and the proportion (right) of repetitive elements in the assembled genome.

Figure 3 .
Figure 3. Genome assembly quality control and contaminant/cobiont detection.(A) BlobPlot of the assembly.Each circle represents a scaffold with its size proportional to the scaffold length.The colour of the circle indicates the taxonomic assignment based on the BLAST similarity search results; (B) ReadCovPlot of the assembly showing the proportion of unmapped and mapped sequences (left panel).The latter is further described according to the rank of the phylum (right panel).

Table 1 .
Summary of the genome and transcriptome sequencing information.Approximately 0.5 g of stored fruit body was ground to powder with liquid nitrogen and used for the construction of an Omni-C library by following the plant tissue protocol for the Dovetail ® Omni-C ® Library Preparation Kit (Dovetail Cat.No. 21005).The ground tissue was transferred to 4 mL 1× PBS and subjected to crosslinking with formaldehyde and digestion with endonuclease DNase I.The quantity and fragment size of the lysate were assessed with Qubit ® Fluorometer and TapeStation D5000 HS ScreenTape, respectively.The qualified lysate was polished at the DNA ends and ligated with biotinylated bridge adaptors, followed by proximity ligation, crosslink reversal of DNA and purification with SPRIselect™ Beads (Beckman Coulter Product No. B23317).The end repair and adapter ligation were performed with the Dovetail™ Library Module for Illumina (Dovetail Cat.No. 21004).The library was then sheared with USER Enzyme Mix and purified with SPRIselect™ Beads.The DNA fragments were isolated in Streptavidin Beads, from which the library was amplified with Universal and Index PCR Primers from the Dovetail™ Primer Set for Illumina (Dovetail Cat.No. 25005).Size selection, targeting fragment sizes between 350 bp and 1000 bp, was performed with SPRIselect™ Beads.The quantity and fragment size of the library were assessed by Qubit ® Fluorometer and TapeStation D5000 HS ScreenTape, respectively.The resulting library was sequenced on an Illumina HiSeq-PE150 platform.Details of the resulting sequencing data are listed in Table

Table 2 .
Genome statistics and sequencing information.

Table 3 .
Information on scaffold names and lengths.

Table 4 .
Summary of the GenomeScope statistics.

Table 5 .
Summary of the TE annotations.