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 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.

Automated summary

The MBD-mediated enrichment method greatly aids in enriching phytoplasma DNA from infected plants for genome sequencing, with a 10- to 30-fold enrichment compared to total DNA. Using this method, most genome regions of phytoplasmas can be sequenced by only one million sequence reads, facilitating the draft genome sequencing of phytoplasmas.