title: Generation and analysis of draft sequences of 'stolbur' phytoplasma from multiple displacement amplification templates.
abstract: Phytoplasma-associated diseases are reported for more than 1,000 plant species worldwide. Only a few genome sequences are available in contrast to the economical importance of these bacterial pathogens. A new strategy was used to retrieve phytoplasma strain-specific genome data. Multiple displacement amplification was performed on DNA obtained from <3 g of plant tissue from tobacco and parsley samples infected with 'stolbur' strains. Random hexamers and Phi29 polymerase were evaluated with and without supplementation by group-assigned oligonucleotides providing templates for Illumina's sequencing approach. Metagenomic drafts derived from individual and pooled strain-specific de novo assemblies were analyzed. Supplementation of the Phi29 reaction with the group-assigned oligonucleotides resulted in an about 2-fold enrichment of the percentage of phytoplasma-assigned reads and thereby improved assembly results. The obtained genomic drafts represent the largest datasets available from 'stolbur' phytoplasmas. Sequences of the two strains (558 kb, 448 proteins and 516 kb, 346 proteins, respectively) were annotated allowing the identification of prominent membrane proteins and reconstruction of core pathways. Analysis of a putative truncated sucrose phosphorylase provides hints on sugar degradation. Furthermore, it is shown that drafts obtained from repetitive-rich genomes allow only limited analysis on multicopy regions and genome completeness.
authors.affiliation: Laboratory of Applied Phytopathology, Institute of Pesticides and Environmental Protection, Belgrade, Serbia.
authors.forename: Jelena
authors.initials: J
authors.lastname: Mitrović
authors.forename: Christin
authors.initials: C
authors.lastname: Siewert
authors.forename: Bojan
authors.initials: B
authors.lastname: Duduk
authors.forename: Jochen
authors.initials: J
authors.lastname: Hecht
authors.forename: Karin
authors.initials: K
authors.lastname: Mölling
authors.forename: Felix
authors.initials: F
authors.lastname: Broecker
authors.forename: Peter
authors.initials: P
authors.lastname: Beyerlein
authors.forename: Carmen
authors.initials: C
authors.lastname: Büttner
authors.forename: Assunta
authors.initials: A
authors.lastname: Bertaccini
authors.forename: Michael
authors.initials: M
authors.lastname: Kube
mesh_terms: D004269:DNA, Bacterial / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D016680:Genome, Bacterial; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques / Q000379:methods; D028528:Petroselinum / Q000382:microbiology; D045582:Phytoplasma / Q000235:genetics* / Q000302:isolation & purification; D017422:Sequence Analysis, DNA; D014026:Nicotiana / Q000382:microbiology
chemical_list: D004269:DNA, Bacterial