Location: National Clonal Germplasm RepositoryTitle: Multiplexed Fragaria Chloroplast Genome Sequencing Author
Submitted to: Acta Horticulturae
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/1/2009
Publication Date: 4/1/2010
Citation: Njuguna, W., Liston, A., Cronn, R., Bassil, N.V. 2010. Multiplexed Fragaria Chloroplast Genome Sequencing. Acta Horticulturae. 859:315-320. Interpretive Summary: Relationships among strawberry species have not been clearly described in the past using visible plant traits or DNA-based molecular tools. The chloroplast, a major organ in plant cells used by the plant to produce food (sugars) contains genetic material (DNA). This DNA from chloroplasts is commonly used for species relationship studies in plants because it is small in size and usually only inherited from the mother. New DNA sequencing technology generally termed, ‘ultra high throughput sequencing’ (Illumina, San Diego, CA), was tested for generating DNA sequence information for the full length of chloroplast DNA in 17 strawberry species. This method resulted in sequencing 76-82% of the chloroplast DNA from the 17 strawberry species. Analysis of these chloroplast DNA sequences from the different strawberry species may show relationships among strawberry species and might allow development of additional tools for species identification.
Technical Abstract: A method to sequence multiple chloroplast genomes that uses the sequencing depth of ultra high throughput sequencing technologies was recently described. Sequencing complete chloroplast genomes can resolve phylogenetic relationships at low taxonomic levels and identify point mutations and indels that can be used in species identification. The objective of this study was to demonstrate multiplex sequencing of Fragaria chloroplast genomes using the Illumina Genome Analyzer. Sixty three PCR fragments averaging 3kb in size were sequenced in four multiplex experiments comprising five or six PCR pools for a total of 22 species. A combination of de novo and reference guided assembly was used to determine the plastome sequences. The plastome used for reference guided assembly was obtained from microread sequencing of F. vesca cv. Hawaii 4. The de novo assembly resulted in plastome coverage of 43-74%. Reference guided assembly (with the program RGA) using species specific references that integrated de novo sequences and the Hawaii 4 reference, increased the estimated plastome coverage to 76-82%. The assembled plastomes were aligned using MAFFT and phylogenetic relationships estimated with parsimony and maximum likelihood. Most nodes were resolved with high bootstrap support, resulting in the first well-resolved chloroplast-based phylogeny of Fragaria.