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Abstract Detail



Systematics Section/ASPT

Ballard Jr, Harvey [1].

Microsatellites and microsatellite-straddling regions from 454 shotgun sequences: Principles, process, and progress in clubmosses (Lycopodiella) and violets (Viola).

Widespread access to affordable genome-scale high-throughput sequencing has made possible new genetic applications and also revolutionized existing approaches, including isolation of microsatellite loci and their flanking regions for non-model organisms. We utilized one recently published approach to identify microsatellite regions in three "reference" species belonging to groups currently under study in our lab, a clubmoss (Lycopodiella subappressa) and two violets (Viola pallens in the allotetraploid "stemless whites", subsect. Stolonosae; and Viola sororia in the allodecaploid "stemless blues", subsect. Boreali-Americanae). We submitted genomic extracts to shotgun genome sequencing and processed the collective 155,000 reads through QDD software. Considering only dinucleotide repeats of at least 8 bp and tri- to heptanucleotide repeats of at least 5 bp as most likely to express population-level variation, we identified 45, 137 and 598 potential microsatellites in Lycopodiella, Viola pallens and Viola sororia, respectively. Sequencing cost ca. $1250 per taxon, personal time was limited to DNA extraction and quantification, and processing of the FASTA file with QDD was easy and rapid. Screening of primer pairs for several di-, tri- and tetranucleotide microsatellites have yielded a very high rate of clean amplification and many reveal substantial allelic variation on regular and high-resolution Metaphor agarose gels, in the reference and related species of the same groups. In the "stemless blue" group, some loci examined from populations of several species show variation in apparently one amplified homoeolog while other loci amplify 2-3 apparent homoeologs, each with allelic variants and expressing species-diagnostic multi-locus patterns. Putative morphological hybrid individuals express additivity or combinations of species-specific fragments and alleles from the prospective parent species at each site. We have also begun investigating the phylogenetic utility of "microsatellite-straddling regions" as a new source of phylogenetically informative low-copy nuclear regions for species-level studies. We have designed primer pairs to amplify larger, 400-800 bp fragments containing a microsatellite using the QDD results from the shotgun sequences, and have successfully amplified various species in Lycopodiella and Viola. We are presently sequencing the amplified fragments, and preliminary trials show some promising species-level variation in certain microsatellite-straddling regions. While Sanger sequencing typically produces degenerating sequence beyond the tandem repeat region in each direction, forming a clear contig from sequence of both directions is reliable in every case, and the microsatellite area itself can be excluded prior to further analysis.

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1 - Ohio University, Environmental & Plant Biology/Molecular & Cellular Bio Program, 315 Porter Hall, Athens, OH, 45701-2979, USA

Keywords:
nuclear microsatellite
next generation sequencing.

Presentation Type: Oral Paper:Papers for Sections
Session: 18
Location: Magnolia/Riverside Hilton
Date: Tuesday, July 30th, 2013
Time: 8:45 AM
Number: 18002
Abstract ID:373
Candidate for Awards:None


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