Abstract
In the present work, a highly pathogenic isolate of Fusarium oxysporum f. sp. lini, which is the most harmful pathogen affecting flax (Linum usitatissimum L.), was sequenced for the first time. To achieve a high-quality genome assembly, we used the combination of two sequencing platforms – Oxford Nanopore Technologies (MinION system) with long noisy reads and Illumina (HiSeq 2500 instrument) with short accurate reads. Given the quality of DNA is crucial for Nanopore sequencing, we developed the protocol for extraction of pure high-molecular-weight DNA from fungi. Sequencing of DNA extracted using this protocol allowed us to obtain about 85x genome coverage with long (N50 = 29 kb) MinION reads and 30x coverage with 2 × 250 bp HiSeq reads. Several tools were developed for genome assembly; however, they provide different results depending on genome complexity, sequencing data volume, read length and quality. We benchmarked the most requested assemblers (Canu, Flye, Shasta, wtdbg2, and MaSuRCA), Nanopore polishers (Medaka and Racon), and Illumina polishers (Pilon and POLCA) on our sequencing data. The assembly performed with Canu and polished with Medaka and POLCA was considered the most full and accurate. After further elimination of redundant contigs using Purge Haplotigs, we achieved a high-quality genome of F. oxysporum f. sp. lini with a total length of 59 Mb, N50 of 3.3 Mb, and 99.5% completeness according to BUSCO. We also obtained a complete circular mitochondrial genome with a length of 38.7 kb. The achieved assembly expands studies on F. oxysporum and plant-pathogen interaction in flax.
Highlights
IntroductionFlax seeds are rich in healthy alpha-linolenic acid (omega-3), lignans, and soluble dietary fibers
Flax (Linum usitatissimum L.) is widely used for the production of seeds and fiber
The purity of DNA is crucial for Nanopore sequencing, so the protocol was developed to extract DNA of sufficient purity from Fusarium fungi
Summary
Flax seeds are rich in healthy alpha-linolenic acid (omega-3), lignans, and soluble dietary fibers They are of great medicinal and nutraceutical value (Goyal et al, 2014; Kezimana et al, 2018; Cullis, 2019) and are potential functional animal feed for improving reproductive capacity and quality of meat, eggs, and milk (Yi et al, 2014; Dutra et al, 2019; Head et al, 2019; Isenberg et al, 2019; Marino et al, 2019; Mattioli et al, 2019). The pathogen is soil-borne, and infection occurs mainly through the roots It spreads inside the vascular tissues, causing water and nutrient blocking, and eventually leading to wilting, yellowing and browning of top parts of the plants, and death (Nair and Kommedahl, 1957; Kroes et al, 1998; Michielse and Rep, 2009). In the present study, using the combination of Oxford Nanopore Technologies (ONT) and Illumina platforms, a highly pathogenic isolate of F. oxysporum f. sp. lini, the most harmful pathogen of flax, was sequenced for the first time
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