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. 2022 Jan 28;14(1):evac010. doi: 10.1093/gbe/evac010

A Highly Contiguous Genome Assembly of Arthrinium puccinoides

Trine Sørensen 1, Celine Petersen 1, Lavinia I Fechete 1, Kåre L Nielsen 1,✉, Teis E Sondergaard 1,✉
Editor: Li-Jun Ma
PMCID: PMC8808539  PMID: 35104337

Abstract

The phylogenetic relationship of the Arthrinium genus has changed throughout the years. For many years, the Arthrinium genus included the Apiospora genus as well. New evidence has now showed that these two genera in fact are phylogenetically different and belong to two different clades. Here, we present the first genome draft within the Arthrinium genus. This genome was sequenced using the MinION platform from Oxford Nanopore Technologies and the assembly was contiguous. The assembly comprises ten contigs totaling 39.8 Mb with an N50 length of 7.9. In the assembly, 11,602 genes were predicted whereof 10,784 were functionally annotated. A total of 37 rRNA genes were observed in the assembly and repeat elements spanning 7.39% of the genome were found. A total of 99 secondary metabolite gene clusters were predicted, showing a high potential of novel secondary metabolites. This genome sequence will not only be useful for further investigation of the Arthrinium clade, but also for discovery of novel secondary metabolite compounds that could be of high interest for the food, agricultural, or pharmaceutical industry.

Keywords: MinION, de novo assembly, genome annotation, secondary metabolites, phylogenetics

Significance

The fungal clade of Arthrinium, which mainly is observed in temperate, cold, and alpine areas, was seen as one group containing not only the Arthrinium genus, but also the genus of Apiospora. Recent evidence, however, separate these two clades in to two different new clades as they were shown to be phylogenetic different. No genomes of this new Arthrinium clade are currently available. In this work, we present the first available genome draft of an Artirinium sp. Our results support the recently reported separation of the current Arthrinium clade into the two new clades—Arthrinium and Apiospora.

Introduction

The genus Arthrinium has had a schizophrenic existence located in different genera as time has passed. Arthrinium is mainly found in temperate, cold, and alpine areas (Pintos and Alvarado 2021), which was also the case for Arthrinium caricicola that is the first proposed and validated Arthrinium (Kunze and Schmidt 1817; Fries 1832). Due to different conidial shape, some Arthrinium spp. were considered to belong to other genera, such as Camptoum, Gonatosporium, Goniosporium, and Sporophleum (Willdenow 1824), but later again associated with Arthrinium (Höhnel 1925; Cooke 1954). Other different genera synonymized with Arthrinium are described in literature (Johnston and Stevenson 1917; Subramanian 1956; Hughes 1953; Cole and Samson 1979; Minter 1985). Notably, one of these is the association with Apiospora through Papularia Fr. because it is the asexual morph of Apiospora. Hence, the biological relation between Arthrinium and Apiospora was established (Saccardo 1875; Höhnel 1919; Petrak 1925; Hudson 1960, 1963; Ellis 1965). Subsequently, other genera were shown to have a biological relation to Apiospora and therefore likewise considered as synonyms of Arthrinium (Liu et al. 1976; Dyko and Sutton 1979; Samuels et al. 1981; Sivanesan 1983; Kirk 1986).

With the use of genetic data, Hyde et al. (1998), placed Arthrinium and Apiospora in their own family named Apiosporaceae. However, Crous and Groenewald (2013), simply suggest Arthrinium as genus name for them both based on the one fungus = one name policy. Both Crous and Groenewald (2013) and Wang et al. (2018) genetically identified multiple species of Arthrinium (now known as Apiospora). However, when Pintos et al. (2019), published marker genes from Arthrinium cariciola, Arthrinium curvatum, and Arthrinium sporophleum, they noted that these Arthrinium together with Arthrinium japonicum and Arthrinium puccinioides clustered in a clade separately from all other Arthrinium (now known as Apiospora). Thus, they proposed that Apiospora and Arthrinium could be phylogenetically different, but that further data were needed (Pintos et al. 2019). Shortly thereafter, the phylogenetic delimitation of Arthrinium and Apoisopra was confirmed by Pintos and Alvarado (2021).

The four Arthrinium genomes available in NCBI (Apiospora malaysianum [ASM650811v1], Apiospora KUC21332 [ASM1716395v1], Apiospora saccharicola [ASM1900006v1], and Apiospora phaeospermum [ASM650353v1]) are in fact Apiospora according to Pintos and Alvarado (2021), meaning that no genome draft is published for Arthrinium. We therefore present the first genome draft of an Arthrinium spp. and hereby give a genetic resource useful for genomic and evolutionary studies. This genome draft also gives insight into the biosynthesis potential of secondary metabolites of this genus which could be of great interest for discovery of novel compounds. We furthermore support the finding of Pintos and Alvarado (2021), through phylogenetic relation between Arthrinium and Apoispora.

Results and Discussion

Genome Sequencing and Assembly

A total of 3,325,171,178 bases, across 211,994 reads, were generated during Oxford Nanopore Technologies sequencing. Of these, 2,794,209,936 bases and 106,349 reads were used in the de novo assembly of Arthrinium puccinoides after quality filtering. The N50 of the read length and the mean read quality were 31,689 bases and 12 Phred score, respectively.

The de novo assembly resulted in a draft genome size of 39.8 Mb spanning 10 contigs (table 1). N50 of the assembly length is 7.9 Mb whereas N99 is 2.3 Mb, suggesting a highly contiguous assembly. Furthermore, Benchmarking Universal Single-Copy Orthologs (BUSCO) was used to assess the assembly completeness in gene space and 97.6% complete BUSCOs were observed, indicating an almost complete gene content. A low observed number of duplicated and fragmented BUSCOs further support a high-quality assembly.

Table 1.

Assembly and Genome Annotation Statistics

Genome Characteristics Value
Assembly Draft genome size (Mb) 39.8
Coverage (×) 70.2
Number of contigs 10
Longest contig (Mb) 9.5
N50 (Mb) 7.9
N99 (Mb) 2.3
GC (%) 53
BUSCO Complete BUSCOs (%) 97.6
Complete and single-copy BUSCOs (%) 97.3
Complete and duplicated BUSCOs (%) 0.3
Fragmented BUSCOs (%) 0.8
Missing BUSCOs (%) 1.6
Annotation Number of predicted genes  11,602
Number of predicted genes with a function 10,784
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Genome Annotation and Repeat Elements

Annotation of the genome resulted in the identification of 11,602 protein-coding genes of which 10,784 genes (92.95%) could be assigned a function bioinformatically (table 1). Noncoding rRNA genes and tRNA genes were predicted across the genome comprising 37 rRNA genes and 174 tRNA genes. Repetitive elements were furthermore observed to span 7.39% of the genome where 1.73% and 1.86% of these were identified as tandem repeats and long-terminal repeat retrotransposons, respectively. Long-interspersed elements account for 0.31% and short-interspersed elements account for 0.13%.

Comparative Genome Analysis

Phylogenetic trees based on whole-genome alignment and alignment of orthogroups were constructed (fig. 1A) to support the phylogenetic delimitation of Arthrinium and Apiospora as proposed by Pintos and Alvarado (2021). The four Apiospora spp. previously stated as Arthrinium spp. (Apiospora malaysianum [ASM650811v1], Apiospora KUC21332 [ASM1716395v1], Apiospora saccharicola [ASM1900006v1], and Apiospora phaeospermum [ASM650353v1]) forms in both cases a separated cluster from Arthrinium puccinoides supporting the phylogenetic delimitation made with marker genes by Pintos and Alvarado (2021). Notably, the two phylogenetic trees are similar even though the phylogenetic tree based on whole-genome alignment contains both genetic and intronic information whereas the tree based orthogroups contains genetic information only.

Fig. 1.

Fig. 1.

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(A) The phylogenetic relation between Apiospora spp. and Arthrinium puccinoides. The Apiospora spp. were formerly known as Arthrinium spp. Fusarium graminearum (ASM24013v3) and Fusarium oxysporum (ASM1308505v1) were included as well. The phylograms were rooted with Aspergillus niger (ASM1928827v1) and Aspergillus oryzae (ASM1340704v2). A phylogenetic relationship based on whole-genome alignment is showed on the left, whereas a phylogenetic relationship based on alignment of 5,057 orthogroups is showed on the right. (B) Number of each type of predicted secondary metabolite genes in the nine different genomes using AntiSMASH.

Secondary Metabolite Potential

Arthrinium puccinoides shows high capacity for synthesizing numerous novel secondary metabolites (fig. 1B). A total of 99 secondary metabolite genes were predicted in the genome draft assembly, showing the highest number of predicted secondary metabolite genes compared with the other genome draft assemblies include in this report. Variants of different classes were predicted: 50 to be polyketide synthases, 16 to be nonribosomal peptide synthetases, 15 to be terpenes, and six to be indoles. Only a few of the secondary metabolite gene clusters could be predicted to code for a known secondary metabolite. With this in mind and the newly delimitation of the two clades (Arthrinium and Apiospora), it opens up for a whole new cluster of fungus to investigate and potential make discovery of novel compounds of high potential from this clade.

Materials and Methods

Growth Conditions, DNA Extraction, and Genome Sequencing

Arthrinium puccinoides (CBS 549.86) was obtained from the Westerdijk Fungal Biodiversity Institute (Utrecht, The Netherlands). The fungus was cultivated on solid Yeast Peptone Glucose (YPG) (10 g/l yeast extract, 20 g/l peptone, and 25 g/l glucose) at 25°C for 5 days, and transferred to liquid YPG media and grown in a rotary shaker for 4 days at 25°C and 100 rpm. The biomass was filtered using a microfilter, followed by lyophilization and subsequently grounded in a mortar.

Genomic DNA was extracted from the lyophilized and grounded mycelium using the Genomic Buffer Set and the Genomic-tips 20/G according to the manufacturer’s protocol. The quality and quantity of the extracted DNA were evaluated using NanoDrop One (ThermoFisher), Qubit 3.0 (Invitrogen) with Qubit dsDNA HS Assay Kit, and 2200 TapeStation (Agilent) with Genomic DNA ScreenTape Analysis according to the manufacturer’s instructions.

A library was constructed using the Genomic DNA by ligation (SQK-LSK109) protocol from Oxford Nanopore Technologies (Oxford, UK) and sequenced on a R9.4.1 flowcell.

De Novo Assembly and Genome Annotation

The raw data was basecalled using Guppy version 3.4.5 (Oxford Nanopore Technologies 2021) in GPU mode using the dna_r9.4.1_450bps_hac.cfg model. The reads were filtered using Filtlong version 0.2.0 (Wick 2018) to a minimum length of 10 kb and a minimum basecall quality of 80 (Q7). Minimap2 version 2.17 (Li 2018) and Miniasm version 0.3 (Li 2016) were used to create the assembly, which subsequently were polished using Racon version 1.3.3 (Vaser et al. 2017) with default settings and two rounds of Medaka version 1.0.1 (Oxford Nanopore Technologies 2018) with default settings. The completeness was assessed with BUSCO version 3.0.2 (Seppey et al. 2019) using the Ascomycota BUSCO data set.

The genome was annotated using AUGUSTUS version 3.4.0 (Stanke et al. 2004) and InterProScan version 5.38-76.0 (Jones et al. 2014) was used for functional annotation with default settings and the following databases MobiDBLite, Pfam, CDD, ProSiteProfiles, PANTHER, Gene3D, ProSitePatterns, PRINTS, SUPERFAMILY, TIGRFAM, SMART, Coils, PIRSF, Hamap, and SFLD. Noncoding RNA genes were predicted using Barrnap version 0.9 (Seemann 2018) and tRNA-scan version 2.0.5 (Chan and Lowe 2019). Repeat sequences were identified using RepeatMasker version 4.1.2 (Tarailo-Graovac and Chen 2009) with eukaryote as species.

Comparative Genome Analysis and Prediction of Secondary Metabolite Genes

Eight genomes were downloaded from NCBI (Apiospora malaysianum [ASM650811v1], Apiospora KUC21332 [ASM1716395v1], Apiospora saccharicola [ASM1900006v1], Apiospora phaeospermum [ASM650353v1]), Fusarium graminearum (ASM24013v3), Fusarium oxysporum (ASM1308505v1), Aspergillus oryzae (ASM1928827v1), and Aspergillus niger (ASM1340704v2). Whole-genome alignment based on entire genomes in nucleotide space of the eight genomes and A.puccinoides genome was generated in CLC Genomics Workbench version 20.0 (Qiagen, Århus) using default setting. The whole-genome alignment was used as input to create an average nucleotide identity comparison with default setting in CLC Genomics Workbench. Afterwards a neighbor joining tree was constructed from the average nucleotide identity comparison in CLC Genomics Workbench. OrthoFinder (Emms and Kelly 2019) was used to identify orthogroups with default settings. Afterwards, OrthoFinder was used to infer a rooted species tree from 5,057 orthogroups using default settings (Emms and Kelly 2017, 2018). The trees was visualized using R version 4.1.1 in RStudio version 2021.09.0 (R Studio Team 2020) using ggtree 3.0.4 (Yu et al. 2016). Secondary metabolite genes were predicted using AntiSMASH version 5.1 (Blin et al. 2019) and visualized using R version 4.1.1 in RStudio version 2021.09.0 (R Studio Team 2020) using ggplot2 3.3.5 (Wickham 2016).

Acknowledgments

This work was supported by the Novo Nordisk Foundation (Grant No. NNF18OC0034952). The authors thank Elsa Sverrisdóttir for helpful comments on the manuscript.

Author Contributions

T.S.: Conceptualization, methodology, investigation, writing—original draft, writing—review and editing, visualization; C.P.: Conceptualization, methodology, investigation, writing—original draft, writing—review and editing, visualization; L.F.: Investigation; K.L.N.: Conceptualization, supervision, funding acquisition, methodology, review and editing; T.E.S.: Conceptualization, supervision, funding acquisition, methodology, review and editing. All authors have read and agreed to the published version of the manuscript.

Data Availability

The raw reads, the assembly, and the annotation can be found at NCBI BioProject PRJNA764836 with the GenBank accession number JAIUNF000000000 for the assembly and the SRA accession number PRJNA764836 for the raw reads.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The raw reads, the assembly, and the annotation can be found at NCBI BioProject PRJNA764836 with the GenBank accession number JAIUNF000000000 for the assembly and the SRA accession number PRJNA764836 for the raw reads.

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