Complete Genome Sequence of “Candidatus Phytoplasma asteris” RP166, a Plant Pathogen Associated with Rapeseed Phyllody Disease in Poland

The complete genome sequence of “Candidatus Phytoplasma asteris” RP166, which consists of one 829,546-bp circular chromosome, is presented in this work. This bacterium is associated with rapeseed phyllody disease in Poland and belongs to the 16SrI-B (i.e., aster yellows) group.

P hytoplasmas are plant pathogens transmitted by phloem-feeding insects from the order Hemiptera. Rapeseed (Brassica napus L.) crops are persistently threatened by "Candidatus Phytoplasma asteris" (aster yellows group, 16SrI-B subgroup), which causes phyllody diseases (1,2). Phyllodies are leafy structures that develop in place of flowers and transform the infected plants into sterile "zombies" (i.e., plants that serve only for phytoplasma propagation). These dramatic morphological changes are induced by bacterial effectors (3). To better study the epidemiology of this disease, genomic resources are in great demand for the investigation of pathogenicity genes and the development of molecular markers.
Strain RP166 was collected from a naturally infected winter rapeseed plant at the Field Experimental Station of the Institute of Plant Protection-National Research Institute (Winna Góra, Poland; coordinates, 52.208921, 17.437842). Healthy Macrosteles laevis leafhoppers (Cicadellidae) were allowed to feed on the infected plant to acquire the bacteria. Next, the insects were maintained on healthy barley for several weeks to increase the phytoplasma titers prior to DNA extraction.
Two platforms were used for shotgun sequencing. For Illumina, a cetyltrimethylammonium bromide (CTAB) buffer protocol (4) was used for DNA extraction, followed by the use of a KAPA library preparation kit (catalog number KK8234) and Invitrogen SizeSelect gels (catalog number G6610-02) for ϳ600-bp fragments. The MiSeq 2 ϫ 300-bp paired-end sequencing (v3 chemistry) produced ϳ10 Gb raw reads. For Oxford Nanopore Technologies (ONT), DNA was prepared using the Illustra Nucleon Phytopure kit (catalog number RPN8510) according to Wouters et al. (5). The library was prepared using the ONT ligation kit (catalog number SQK-LSK109) without shearing or size selection. The MinION run (R9.4 chemistry) produced 1,697,567 raw reads (ϳ5.9 Gb; N 50 , 12,607 bp). Guppy v2.3.1 was used for base calling with a minimum quality score of 7; no further processing of the ONT reads was conducted.
Strain RP166 has one 829,546-bp circular chromosome with 27.7% GϩC content; no plasmids were found. The Illumina and ONT reads provided 116ϫ and 597ϫ coverage, respectively. The annotation contains 6 rRNA genes, 32 tRNA genes, 753 protein-coding genes, and 69 pseudogenes.
Data availability. The raw reads have been deposited at the NCBI Sequence Read Archive under the accession numbers SRR12000858 and SRR12000859. The genome sequence has been deposited at GenBank/ENA/DDBJ under the accession number CP055264.

ACKNOWLEDGMENTS
The Illumina sequencing service was provided by the Earlham Institute (Norwich, UK). The funding for this project was provided by the Human Frontier Science Program (RGP 0024/2015) to S.A.H. and A.Z., with additional support from the BBSRC Institute Strategy Programme (BB/P012574/1) and the John Innes Foundation, and by Academia Sinica and the Ministry of Science and Technology of Taiwan (MOST 106-2311-B-001-028-MY3) to C.-H.K. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.