Phytoplasma have resisted all attempts of cell-free cultivation so far. This problem hampers genome research. Elaborate and material intensive approaches are used to enrich the phytoplasma DNA. We present an amplification-based approach to obtain phytoplasma DNA from a few grams of plant tissue for downstream applications such as genomic draft sequencing. Total DNA was extracted by CTAB extraction from tobacco and parsley infected by stolbur strains 284/09 and 231/09, respectively. For enrichment, genomic DNA was amplified using oligonucleotides deduced from the four published complete phytoplasma genomes, random hexamers and Phi29 polymerase. Twenty-eight oligonucleotides were selected by frequency and distribution in the complete genomes. In additional experiments, the application of phytoplasma-specific primers P1/P7 was evaluated. De novo assemblies of short-reads with a length of 36 bases were generated. BLASTX against NCBI’s NRPROT database using contigs with a minlength of at least 300 bp was analysed with MEGAN and taxonomical assignment of the contigs performed. Enrichment was measured by illumina’s sequencing by synthesis approach. Up to a fifteen-fold increase of obtained phytoplasma draft sequence resulted from the usage of the determined oligonucleotides. Individual assemblies of short single-reads resulted in an average contig length of 1.3 kb for strain 231/09 and 2.5 kb for strain 284/09 and a total contig length of >474 kb and >498 kb, respectively. Preliminary, results indicate only weak phytoplasma enrichment in the amplification experiments supplemented with P1 and P7 oligonucleotides. Combining the reads of the individual experiments resulted in a draft sequence of >516kb for strain 231/09 and >557 kb for strain 284/09. Sequencing and annotation results highlight the potential of this strategy for uncharacterized phytoplasma genomes in particular. It is shown that cost-saving short-read sequencing can be used to generate efficient draft sequences from these templates.
Enrichment of phytoplasma DNA by selected oligonucleotides and phi29 polymerase amplification / Siewert C.; J. Mitrovic; B. Duduk; J. Hecht; K. Mölling; F. Bröcker; S. von Bargen; A. Bertaccini; M. Kube. - STAMPA. - (2012), pp. 100-100. (Intervento presentato al convegno XXIInd International Conference on Virus other Graft Transmissible Diseases of Fruit Crops tenutosi a Rome, Italy nel June 3-8, 2012).
Enrichment of phytoplasma DNA by selected oligonucleotides and phi29 polymerase amplification.
DUDUK, BOJAN;BERTACCINI, ASSUNTA;
2012
Abstract
Phytoplasma have resisted all attempts of cell-free cultivation so far. This problem hampers genome research. Elaborate and material intensive approaches are used to enrich the phytoplasma DNA. We present an amplification-based approach to obtain phytoplasma DNA from a few grams of plant tissue for downstream applications such as genomic draft sequencing. Total DNA was extracted by CTAB extraction from tobacco and parsley infected by stolbur strains 284/09 and 231/09, respectively. For enrichment, genomic DNA was amplified using oligonucleotides deduced from the four published complete phytoplasma genomes, random hexamers and Phi29 polymerase. Twenty-eight oligonucleotides were selected by frequency and distribution in the complete genomes. In additional experiments, the application of phytoplasma-specific primers P1/P7 was evaluated. De novo assemblies of short-reads with a length of 36 bases were generated. BLASTX against NCBI’s NRPROT database using contigs with a minlength of at least 300 bp was analysed with MEGAN and taxonomical assignment of the contigs performed. Enrichment was measured by illumina’s sequencing by synthesis approach. Up to a fifteen-fold increase of obtained phytoplasma draft sequence resulted from the usage of the determined oligonucleotides. Individual assemblies of short single-reads resulted in an average contig length of 1.3 kb for strain 231/09 and 2.5 kb for strain 284/09 and a total contig length of >474 kb and >498 kb, respectively. Preliminary, results indicate only weak phytoplasma enrichment in the amplification experiments supplemented with P1 and P7 oligonucleotides. Combining the reads of the individual experiments resulted in a draft sequence of >516kb for strain 231/09 and >557 kb for strain 284/09. Sequencing and annotation results highlight the potential of this strategy for uncharacterized phytoplasma genomes in particular. It is shown that cost-saving short-read sequencing can be used to generate efficient draft sequences from these templates.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
