ABSTRACT
We sequenced and annotated the genomes of the ascomycete fungi Trichoderma harzianum, Trichoderma aggressivum f. europaeum, and Purpureocillium lilacinum. Moreover, we developed a website to allow users to interactively analyze the assemblies, gene predictions, and functional annotations of these species and 70+ previously sequenced fungi.
KEYWORDS: fungal genomics, ascomycetes, mycoparasites
ANNOUNCEMENT
Trichoderma harzianum and Trichoderma aggressivum are mycoparasites that can cause green mold disease on mushroom-forming fungi, including the white button mushroom Agaricus bisporus (1). Phylogenetically, they are members of the Harzianum/Virens clade of the Trichoderma species complex (2). The species T. aggressivum encompasses the biotypes aggressivum and europaeum, which originate from North America and Europe, respectively (3). The ascomycete P. lilacinum is a ubiquitous saprotroph, found in soil, dead organic matter, insects, and air (4). We serendipitously discovered that it can inhibit growth of the mushroom-forming fungus Schizophyllum commune.
T. harzianum CBS 354.33, originally isolated from soil, and T. aggressivum CBS 100526, originally isolated from mushroom compost, were obtained from the Westerdijk Institute. P. lilacinum was isolated from the air and deposited in the Westerdijk collection with accession number CBS 150709. A spore suspension was incubated for 3 days at 30°C in liquid culture in Schizophyllum commune minimal medium (5). Genomic DNA was extracted and purified from lysed cells using the MagMAX Plant DNA Isolation Kit (Thermo Scientific, USA) and sonicated using a Bioruptor (Diagenode, Belgium) for 10 minutes, with a 30-/90-second on/off cycle on low power and the temperature in the water bath at 4°C, resulting in fragments of approximately 700 bp. The NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs) was used to generate Illumina libraries. These libraries were sequenced by the Utrecht Sequencing Facility (Utrecht; The Netherlands; https://useq.nl) using an Illumina NextSeq2000 sequencer with a 2 × 100 bp protocol. The reads were trimmed on both ends to a quality score of 15 using BBMap version 37.88 (https://sourceforge.net/projects/bbmap/) and assembled with SPAdes version 3.15.5 (6) with kmer lengths 21, 33, 55, 77, and 99 and the parameter “--careful.” Scaffolds shorter than 1,000 bp were removed from the assembly. Genes were predicted with AUGUSTUS version 3.3.2 (7) using the provided parameter set of the related ascomycete Aspergillus nidulans and the settings “--genemodel=complete” and “--noInframeStop=true.” The predicted proteins were functionally annotated as previously described (8). Repetitive sequences were identified with RepeatModeler version 2.0.3 (9).
A website was created with a portal for each genome and for all 70+ genomes that were previously sequenced and published by us (https://fungalgenomics.science.uu.nl/). The genome portals allow users to interactively analyze the assembly, gene predictions, and functional annotations. This includes a JBrowse genome browser (10) with Apollo plugin to allow the manual curation of gene models (11), a Blast function to search for sequence similarity in the assembly and gene predictions (12), and full access to the functional annotations of the proteins.
The sizes of the genome assembly were 38.51, 38.95, and 40.62 Mbp for T. harzianum, T. aggressivum, and P. lilacinum, respectively, and BUSCO completeness scores were 99.66% for both Trichoderma spp. and 98.62% for P. lilacinum, indicating a high quality of assembly and gene prediction for all three species (Table 1). These genome annotations and the genome portals will be a valuable resource to study these three competitors of mushroom-forming fungi.
TABLE 1.
Statistics of the three genome annotations
| Trichoderma harzianum | Trichoderma aggressivum f. europaeum | Purpureocillium lilacinum | |
|---|---|---|---|
| Strain | CBS 354.33 | CBS 100526 | CBS 150709 |
| Sequencing read pairs | 23,531,919 | 18,241,525 | 19,347,672 |
| Version of assembly and annotations | Trihar35433_1 | Triagg1 | Purlil1 |
| Assembly | |||
| Sequences in assembly | 171 | 318 | 928 |
| Coverage | 122× | 93× | 100× |
| Total assembly length (Mbp) | 38.51 | 38.95 | 40.62 |
| Assembly GCa content (%) | 49.3 | 49.06 | 58.33 |
| Assembly gaps (%) | 0 | 0.01 | 0.02 |
| L50 number (#) | 6 | 20 | 25 |
| N50 length (bp) | 2,296,050 | 579,860 | 470,782 |
| Repetitive content (%) | 4.23 | 1.9 | 3.99 |
| Predicted genes | |||
| Genes | 11,326 | 11,015 | 14,434 |
| Gene length (median) | 1,422 | 1,431 | 1,489 |
| Transcript length (median) | 1,281 | 1,281 | 1,296 |
| Exon length (median) | 297 | 298 | 288 |
| CDS length (median) | 1,278 | 1,278 | 1,293 |
| Protein length (median) | 426 | 426 | 431 |
| Spliced genes (total, %) | 8,366 (73.87%) | 8,160 (74.08%) | 10,853 (75.19%) |
| Exons per gene (median) | 2 | 2 | 3 |
| Intron length (median) | 65 | 66 | 65 |
| Introns per spliced gene (median) | 2 | 2 | 2 |
| Gene density (genes/Mbp) | 294.09 | 282.79 | 355.36 |
| Coding content of assembly (bp, %) | 17,783,979 (46.18%) | 17,306,514 (44.43%) | 23,597,817 (58.1%) |
| Functional annotations | |||
| Unique PFAM domains | 4,125 | 4,118 | 4,129 |
| Genes with PFAM domain (total, %) | 8,661 (76.47%) | 8,226 (74.68%) | 8,386 (58.1%) |
| Genes with GO annotation (total, %) | 5,414 (47.8%) | 5,142 (46.68%) | 5,360 (37.13%) |
| Genes with secretion signal (total, %) | 1,053 (9.3%) | 970 (8.81%) | 1,259 (8.72%) |
| Genes annotated as small secreted protein (total, %) | 911 (8.04%) | 991 (9.00%) | 1,146 (7.94%) |
| Genes with transmembrane domain (total, %) | 2,141 (18.9%) | 2,063 (18.73%) | 2,543 (17.62%) |
| Genes annotated as transcription factor (total, %) | 487 (4.3%) | 437 (3.97%) | 497 (3.44%) |
| Genes annotated as protease (total, %) | 477 (4.21%) | 438 (3.98%) | 474 (3.28%) |
| Genes annotated as CAZyme (total, %) | 456 (4.03%) | 430 (3.9%) | 454 (3.15%) |
| Secondary metabolism gene cluster | 54 (0.48%) | 58 (0.53%) | 32 (0.22%) |
| BUSCO2 completeness (%) | 99.66% (single copy: 99.31%, duplicated: 0.34%) | 99.66% (single copy: 99.66%, duplicated: 0.0%) | 98.62% (single copy: 97.93%, duplicated: 0.69%) |
guanine-cytosine.
ACKNOWLEDGMENTS
This publication is part of project number VI.Vidi.192.065 of the VIDI research program which is (partly) financed by the Dutch Research Council (NWO). This project has received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (grant agreement number 716132). We acknowledge the Utrecht Sequencing Facility (USEQ) for providing sequencing service and data. USEQ is subsidized by the University Medical Center Utrecht and The Netherlands X-omics Initiative (NWO project 184.034.019).
Contributor Information
Robin A. Ohm, Email: r.a.ohm@uu.nl.
Jennifer Geddes-McAlister, University of Guelph, Canada.
DATA AVAILABILITY
All data are available under NCBI BioProject PRJNA1034581. The raw sequence reads of T. aggressivum, T. harzianum, and P. lilacinum are deposited in NCBI Sequence Read Archive (SRA) under accession numbers SRR26638389, SRR26638390, and SRR26638391, respectively. The genome assemblies and gene predictions are deposited in NCBI GenBank under accession numbers JAWRVG000000000, JAWRVH000000000, and JAWRVI000000000, respectively. The genome annotations can also be accessed at https://fungalgenomics.science.uu.nl.
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Associated Data
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Data Availability Statement
All data are available under NCBI BioProject PRJNA1034581. The raw sequence reads of T. aggressivum, T. harzianum, and P. lilacinum are deposited in NCBI Sequence Read Archive (SRA) under accession numbers SRR26638389, SRR26638390, and SRR26638391, respectively. The genome assemblies and gene predictions are deposited in NCBI GenBank under accession numbers JAWRVG000000000, JAWRVH000000000, and JAWRVI000000000, respectively. The genome annotations can also be accessed at https://fungalgenomics.science.uu.nl.