Journal
BIOTECHNIQUES
Volume 46, Issue 3, Pages 217-223Publisher
FUTURE SCI LTD
DOI: 10.2144/000113085
Keywords
microsatellites; 454 sequencing; simple sequence repeats; FIASCO; ISSR-PCR
Funding
- Tree Protection Cooperative Programme (TPCP)
- Department of Science and Technology (DST) & National Research Foundation (NRF) Centre of Excellence in Tree Health Biotechnology (CTHB)
- Technology and Human Resources for Industry Programme (THRIP)
- Department of Trade and Industry (DTI) of South Africa
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Robust molecular markers such as microsatellites arc important tools used to understand the dynamics of natural populations, but their identification and development are typically time consuming and labor intensive. The recent emergence of so-called next-generation sequencing raised the question as to whether this new technology might be applied to microsatellite development. Following this view, we considered whether deep sequencing using the 454 Life Sciences/Roche GS-FLX genome sequencing system could lead to a rapid protocol to develop microsatellite primers as markers for genetic studies. For this purpose, genomic DNA was sourced from three unrelated organisms: a fungus (the pine pathogen Fusarium circinatum), an insect (the pine-damaging wasp Sirex noctilio), and the wasp's associated nematode parasite (Deladenus siricidicola). Two methods, FIASCO (fast isolation by AFLP of sequences containing repeats) and ISSR-PCR (inter-simple sequence repeat PCR), were used to generate microsatellite-enriched DNA for the 454 libraries. From the resulting 1.2-1.7 megabases of DNA sequence data, we were able to identify 873 microsatellites that have sufficient flanking sequence available for primer design and potential amplification. This approach to microsatellite discovery was substantially more rapid, effective, and economical than other methods, and this study has shown that pyrosequencing provides an outstanding new technology that can be applied to this purpose.
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