Enrichment of phytoplasma genome DNA through a methyl-CpG binding domain-mediated method for efficient genome sequencing

Phytoplasmas are obligate intracellular phytopathogenic bacteria infecting many agriculturally important plants. Although the information on the genome sequences is very useful, it is difficult to sequence phytoplasma genomes because pure phytoplasma DNA is unavailable. Here, we explored whether a m...

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Bibliographic Details
Published in:Journal of general plant pathology : JGPP 2021-05, Vol.87 (3), p.154-163
Main Authors: Nijo, Takamichi, Iwabuchi, Nozomu, Tokuda, Ryosuke, Suzuki, Takumi, Matsumoto, Oki, Miyazaki, Akio, Maejima, Kensaku, Oshima, Kenro, Namba, Shigetou, Yamaji, Yasuyuki
Format: Article
Language:English
Subjects:
Agriculture
Bacterial and Phytoplasma Diseases
Binding
Biomedical and Life Sciences
CpG islands
Deoxyribonucleic acid
DNA
DNA sequencing
Domains
Enrichment
Genomes
Host plants
Life Sciences
Microbiology
Next-generation sequencing
Nucleotide sequence
Phyllody
Phytoplasma
Plant bacterial diseases
Plant Pathology
Plasmids
rRNA
tRNA
Yellows
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Summary:Phytoplasmas are obligate intracellular phytopathogenic bacteria infecting many agriculturally important plants. Although the information on the genome sequences is very useful, it is difficult to sequence phytoplasma genomes because pure phytoplasma DNA is unavailable. Here, we explored whether a methyl-CpG binding domain (MBD)-mediated enrichment method for bacterial DNA could facilitate enrichment of phytoplasma DNA from infected plants for next-generation sequencing of phytoplasma genome. Quantitative PCR revealed that phytoplasma DNA was enriched 10- to 30-fold by MBD-mediated enrichment compared to total DNA from the infected plant. Using the enriched DNA of the ‘ Candidatus ( Ca .) Phytoplasma asteris’ onion yellows strain (OY-M), most genome regions were sequenced by only one million sequence reads generated by an Illumina MiSeq platform. In addition, we present the first draft genome sequence of the ‘ Ca . P. asteris’ hydrangea phyllody strain (HP) by using MBD-mediated enrichment from total DNA of the natural host plant. The draft features 36 contigs of total length 597,762 bp with 521 coding sequences, one rRNA operon, and 30 tRNA genes. One contig resembles plasmids of ‘ Ca . P. asteris’-related strains. Pairwise alignment with the OY-M genome indicated that most regions of the HP genome except for repetitive regions had been sequenced. Thus, MBD-mediated enrichment greatly aids genome sequencing of phytoplasmas.
ISSN:1345-2630
1610-739X
DOI:10.1007/s10327-021-00993-z