Systematics/Phytogeography / Taxonomie/ Section
Parks, Matthew , Liston, Aaron , Cronn, RC .
Phylogenetic Analysis of Pinus Based on 14 Complete Chloroplast Genomes: a Demonstration of Next-Generation Sequencing Technology.
Chloroplast DNA sequences are widely used in plant systematic and population genetic studies. However, the conservative nature of chloroplast sequence evolution often limits phylogenetic resolution and provides low statistical power in population genetics. To gain maximal access to the evolutionary record contained in gymnosperm chloroplast genomes, we are evaluating multiplex sequencing-by-synthesis (MSBS) of multiple plastomes on the Illumina Genome Analyzer using Solexa sequencing technology. We PCR-amplified the ca. 120 kb plastome of 11 Pinus species and Picea sitchensis using 35 ca. 3.5 kb primer pairs. Sheared amplicon fragments were ligated to modified adapters that included 3 bp multiplexing tags. Samples were then multiplexed at 4 or 5 genomes per lane during sequencing. Tagged microreads from each species were assembled using a combination of de novo and reference-guided assembly using the previously published plastomes from Pinus thunbergii and P. koraiensis as references. Our methods produced 12 draft chloroplast genomes at 78% - 99% completion and averaging 36X to 92X sequencing depth. Estimated error rates in the 11 Pinus species and Picea sitchensis ranged from 0.04 – 0.37% based on a comparison to ca. 5,000 bp of Sanger sequence from 7 cpDNA loci. Critically, whole genome sequences are ~26X larger than these 7 loci but contain 51X more phylogenetic information, suggesting that the whole genome data is twice as informative on a per-nucleotide basis and should be exceptionally useful in phylogenetic analyses and population level studies. Indeed, maximum parsimony analysis using based on the 12 new and 2 previously published chloroplast genomes resulted in 100% bootstrap support for 11 of 12 ingroup nodes.
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1 - Oregon State University, Botany and Plant Pathology, Oregon State University, Corvallis, Oregon, 97331, USA
2 - Oregon State University, Department of Botany & Plant Pathology, 2082 Cordley Hall, Corvallis, Oregon, 97331-2902, USA
3 - USDA Forest Service, Forest Genetics, Pacific Nothwest Research Station, 3200 SW Jefferson Way, Corvallis, Oregon, 97331, USA
multiplex genome sequencing
Solexa sequencing technology.
Presentation Type: Oral Paper:Papers for Sections
Location: Room 6/Woodward
Date: Tuesday, July 29th, 2008
Time: 9:15 AM