Enrichment of phytoplasma genome DNA through a methyl-CpG binding domain-mediated method for efficient genome sequencing
Enrichment of phytoplasma genome DNA through a methyl-CpG binding domain-mediated method for efficient genome sequencing
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DOI：
10.1007/s10327-021-00993-z
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发表时间：
2021-03
影响因子：
1.2
通讯作者：
Takamichi Nijo;Nozomu Iwabuchi;Ryosuke Tokuda;Takumi Suzuki;Oki Matsumoto;Akio Miyazaki;K. Maejima;K. Oshima;S. Namba;Y. Yamaji
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作者：
Takamichi Nijo;Nozomu Iwabuchi;Ryosuke Tokuda;Takumi Suzuki;Oki Matsumoto;Akio Miyazaki;K. Maejima;K. Oshima;S. Namba;Y. Yamaji
植原体是一种专性胞内植物病原细菌，感染许多重要的农业植物。尽管植原体基因组序列的信息非常有用，但由于无法获得纯的植原体DNA，因此难以对植原体基因组进行测序。在这里，我们探讨了甲基-CpG结合域（MBD）介导的富集细菌DNA的方法是否可以促进富集植原体DNA从受感染的植物植原体基因组的下一代测序。定量PCR显示，植物原体DNA富集10- 30倍的MBD介导的富集相比，总DNA从受感染的植物。利用富集的“"DNA，对于紫菀植原体洋葱黄化菌株（OY-M），通过由Illumina MiSeq平台生成的仅一百万个序列读段对大多数基因组区域进行测序。此外，我们提出了第一个草案的基因组序列的'Ca。利用MBD富集法对紫菀绣球菌（P. asteris' Hydrangea phyllody strain，HP）的总DNA进行了初步研究。该草案具有36个重叠群，总长度为597，762 bp，具有521个编码序列，一个rRNA操纵子和30个tRNA基因。一个重叠群类似于'Ca. P. P.与OY-M基因组的成对比对表明，除了重复区域之外，HP基因组的大多数区域都已测序。因此，MBD介导的富集大大有助于植原体的基因组测序。
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.