Cronn, RC , Liston, Aaron .
Multiplex Sequencing of Plant Chloroplast Genomes Using Illumina/Solexa Sequencing-By-Synthesis Technology.
Chloroplast and mitochondrial organellar genomes are widely used in plant germplasm characterization because they offer a simple means to evaluate cytoplasmic diversity, germplasm differentiation, and taxonomic affinities. Due to their haploid nature and (typically) uniparental transmission, these genomes are highly responsive to drift. These positive attributes are counterbalanced by two undesirable features; a large size and highly conservative mutation rate. Haplotype variation – when present – is rarely found in a single mutational ‘hotspot’, but is usually dispersed across the genome in simple repeats, small rearrangements, and single nucleotide polymorphisms. Because of the limited variation detected in most taxa, a host of genes, spacers, introns, and microsatellite repeats are frequently pre-screened to identify “tortoises” and “hares” so that genotyping efforts can be tailored to specific taxonomic questions.
An alternative to this endless pursuit is to sequence entire genomes and evaluate all mutational classes genome-wide. In this presentation, we show how multiplex “sequencing-by-synthesis” (MSBS) on the Illumina Genome Analyzer is one way to achieve this goal. We have successfully used MSBS to sequence PCR-amplified plastomes from 4 to 6 species of Pinus simultaneously. By ‘tagging’ each genome with a unique adapter, microreads (36 - 40 bp) can be sorted and independently assembled using a combination of de novo and reference-guided steps. Results to date show that draft genomes can be rapidly assembled that are 85% to 98% complete, with an average sequencing depth over 50X. The power of this approach is highlighted with Pinus torreyana, a species that has yet to reveal intraspecific cpDNA divergence in previous RFLP and cpSSR studies. In a comparison of two individuals, we identified 5 SNPs in 101 kb of chloroplast DNA. We conclude this talk by considering how MSBS might be applied to population-level screening of additional genes and genomes.
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WS1 Applying Modern Genomic Tools to the Management and Characterization of Plant Genetic Resources
1 - USDA Forest Service, Forest Genetics, Pacific Nothwest Research Station, 3200 SW Jefferson Way, Corvallis, Oregon, 97331, USA
2 - Oregon State University, Department of Botany & Plant Pathology, 2082 Cordley Hall, Corvallis, Oregon, 97331-2902, USA
Presentation Type: Workshop
Location: Blair CD/Gage
Date: Sunday, July 27th, 2008
Time: 2:00 PM