ABSTRACT
We present two whole-genome sequences of Colletotrichum strains which were isolated from Eleusine indica and Echinochloa crus-galli using Nanopore and Illumina technologies, as part of screening for potential mycoherbicide. The genome sequences will provide important genetic information and will be useful for further research into secondary metabolites of Colletotrichum.
KEYWORDS: Colletotrichum, anthracnose, comparative genomics, nanopore sequencing, Illumina, glomerella
ANNOUNCEMENT
Colletotrichum eleusines isolate was obtained from diseased goosegrass plants of Hubei province, China (30°17′24″N, 114°07′56″ E, 30 m above sea level). C. echinochloae isolate was obtained from diseased barnyardgrass plants of Hubei province, China (32°7′49″N, 112°20′35″ E, 91 m above sea level). All strains were isolated through single spore isolation, and their pathogenicity on the host was confirmed by the Koch’s postulates (1, 2). The strains were incubated at 25°C on PDA medium for 7 days and verified by observation of morphology, concatenated ITS, Sod2, Apn2, and Mat1-2 sequences (2).
All isolates used in this study were cultivated in potato dextrose broth for 7 days, and genomic DNA was extracted from fresh mycelial tissue using the CTAB method (3) further purified with the Wizard Genomic DNA purification Kit (Promega, Madison, WI, USA). The high-molecular weight genomic DNA (gDNA) was obtained by removing fragments shorter than 2 kbp using the Agencourt AMPure XP (Beckman Coulter Inc., USA). The gDNA was repaired using the PreCR Repair Mix (New England Biolabs, Ipswich, MA, USA) before subjected to library construction. Both Colletotrichum genomes were sequenced and assembled using a combination of long and short reads. Long reads were generated first by using an Oxford Nanopore Technologies Ligation Sequencing Kit (SQK-LSK109, Oxford Nanopore Technologies, Oxford, UK), and then, a library of each strain was sequenced with primed R9.4 Spot-On Flow cells for 60 h on a PromethION sequencer (Oxford Nanopore Technologies). Short reads were generated using genomic DNA libraries constructed with an NEBNext UltraTM Prep Kit (150-bp paired end reads, with a read length of 500 bp) and sequenced on the Illumina NovaSeq 6000 platform (Illumina Inc., San Diego, California, USA). The Oxford Nanopore Technologies (ONT) reads fastq data were basecalled using Guppy v.3.03 (HAC model). FastQC was used for quality control for Illumina reads. ONT reads with a Q-score lower than 7 were filtered out. The long-read Nanopore sequences were assembled with NECAT v.20200119 (4), and raw reads of ONT were mapped to the draft assembly using minimap2 v.2.11 (5), and two rounds of polishing were performed with Racon v.1.4.11 (6). To further improve assembly, Illumina short reads were mapped to the assembly using BWA v.0.7.17 (7) and two rounds of polishing were performed with Pilon v.1.23 (8). Default parameters were used except where otherwise noted.
The nuclear genome of C. eleusines CCTCC2020688 consisted of 15 contigs with a total assembled length of 53.51 Mbp, 51.11% GC content, and a maximum contig size of 6,664,387 bp (Table 1). In comparison, the nuclear genome assembly of C. echinochloae CCTCC2020689 consisted of 21 contigs with a total assembled length of 62.25 Mbp, 46.59% GC content, and a maximum contig size of 8,328,496 bp. Assembly completeness was assessed with the program BUSCO (v.3.0.1) (9), which estimated the gene complement to be 99.3% complete for both C. eleusines and C. echinochloae genomes, using a 290-gene database (fungi_odb9).
TABLE 1.
Summary statistics of C. eleusines and C. echinochloae genomes
| Statistics | ||
|---|---|---|
| Variables | C. eleusines | C. echinochloae |
| Isolate | CCTCC2020688 | CCTCC2020689 |
| NCBI accession | JADEZO000000000 | JADEZT000000000 |
| Sequencing technology | ONT PromethION & Illumina NovaSeq 6000 | ONT PromethION & Illumina NovaSeq 6000 |
| Genome assembly | ||
| Assembly size (bp) | 53,507,824 | 62,246,358 |
| Number of contigs | 15 | 21 |
| Contig N50 length (bp) | 5,135,075 | 5,346,453 |
| Contig N90 length (bp) | 2,723,754 | 3,463,513 |
| GC (%) | 51.11 | 46.59 |
| Average contig length (bp) | 3,567,188.27 | 2,964,112.29 |
| Longest contig (bp) | 6,664,387 | 8,328,496 |
| Shortest contig (bp) | 33,194 | 25,553 |
| Total reads for ONT (bp) | 48,350,746 | 610,642 |
| Read length N50 for Nanopore (bp) | 25,147 | 20,000 |
| Total reads for Illumina (bp) | 48,350,746 | 55,323,334 |
| Average depth for Illumina | 133.77 | 132.23 |
| Genome completeness | ||
| Complete BUSCOs | 288 | 288 |
| Complete and single BUSCOs | 288 | 288 |
| Complete and duplicated BUSCOs | 0 | 0 |
| Fragmented BUSCOs | 1 | 1 |
| Missing BUSCOs | 1 | 1 |
ACKNOWLEDGMENTS
We are grateful to Dr. Tom Hsiang at the Guelph University for reviewing the paper.
This work was supported by the National Natural Science Foundation of China (No. 31901903) and the Youth Science Foundation of Hubei Academy of Agricultural Sciences (No. 2020NKYJJ09).
Contributor Information
Ruhai Li, Email: ruhaili73@163.com.
Jason E. Stajich, University of California Riverside, USA
DATA AVAILABILITY
The genome sequences described in this article have been deposited in DDBJ/ENA/GenBank under the accession number JADEZO000000000 for C. eleusines CCTCC2020688 (BioProject: PRJNA671855; Biosample: SAMN16552423), [the version described in this paper is the first version (JADEZO000000000.1), and the raw sequencing reads are available under SRA accession numbers SRX9363665 and SRX9363664, respectively] and JADEZT000000000 for C. echinochloae CCTCC2020689 (BioProject: PRJNA672254; Biosample: SAMN16551125) [the version described in this paper is the first version (JADEZT000000000.1), with raw sequencing reads available under SRA accession numbers SRX9369587 and SRX9369586, respectively].
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Associated Data
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Data Availability Statement
The genome sequences described in this article have been deposited in DDBJ/ENA/GenBank under the accession number JADEZO000000000 for C. eleusines CCTCC2020688 (BioProject: PRJNA671855; Biosample: SAMN16552423), [the version described in this paper is the first version (JADEZO000000000.1), and the raw sequencing reads are available under SRA accession numbers SRX9363665 and SRX9363664, respectively] and JADEZT000000000 for C. echinochloae CCTCC2020689 (BioProject: PRJNA672254; Biosample: SAMN16551125) [the version described in this paper is the first version (JADEZT000000000.1), with raw sequencing reads available under SRA accession numbers SRX9369587 and SRX9369586, respectively].