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United States Department of Agriculture

Agricultural Research Service

Research Project: DISCOVERY AND DEVELOPMENT OF NATURAL PRODUCT-BASED WEED MANAGEMENT METHODS

Location: Natural Products Utilization Research

Title: Optimized construction of microsatellite-enriched libraries

Authors
item Techen, Natascha -
item Arias De Ares, Renee
item Pan, Zhiqiang
item Khan, Ikhlas -
item Scheffler, Brian

Submitted to: Molecular Ecology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: November 23, 2009
Publication Date: May 3, 2010
Citation: Techen, N., Arias De Ares, R.S., Pan, Z., Khan, I., Scheffler, B.E. 2010. Optimized construction of microsatellite-enriched libraries. Molecular Ecology. 10:508-515.

Interpretive Summary: Molecular DNA markers can be used for genome-assisted breeding and population genetic studies of animals and plants, fingerprinting, authentication of varieties in the industry of natural products, and detection of human diseases. Such molecular markers, microsatellites or simple sequence repeats (SSR), can be identified by using techniques that are costly, consist of multiple steps, and result in a low number of identified SSRs. We have developed a method that facilitates the isolation of very high numbers of SSRs from genomic libraries that can be used as molecular markers. The method was successfully applied on four organisms, a nematode, a fungus, a crop plant and a tree, to demonstrate that the protocol worked effectively across phyla.

Technical Abstract: The construction of simple sequence repeat (SSR) libraries is an indispensable tool to search for molecular markers as complete genome sequences are still not available for the majority of species of interest. Numerous protocols are available in the literature for the construction of SSR-enriched libraries; however, sometimes their lack of detail and optimization can restrict their efficacy. We have designed and tested various adapters and ligation methods; we also tested oligo-repeat combinations and hybridization temperatures, and created libraries with this new protocol for four organisms: Ipomoea, Chionanthus, Rotylenchus and Puccinia. Presence of repeats on the contigs analyzed was as low as 74% and as high as 210%, indicating that more than one repeat was present per contig. A simplified protocol is provided for the successful generation of SSR-enriched libraries.

Last Modified: 7/10/2014