Microsatellite Discovery using 454 Sequencing in an Endangered North American Hardwood Tree

Butternut (Juglans cinerea L), a native hardwood tree of the eastern U.S., is being devastated by an introduced fungal pathogen.  As part of an investigation of the genetics of disease resistance in this species, we used Roche 454 sequencing to identify microsatellite repeats in butternut and a close relative, Japanese walnut (J. ailantifolia).  Di-, tri-, and tetra-nucleotide repeat sequences were isolated from a genomic DNA pool using the enrichment procedure of Glenn and Schable (2005).  From 67,928 reads in butternut and 40,783 in Japanese walnut, 2653 butternut and 1298 Japanese walnut contigs were assembled.  These we visualized in Tablet (Milne et al. 2010).  Primers were designed for quality marker candidates based on the appearance of microsatellite repeats and polymorphisms in the contigs.  After sequencing we submitted these sequences to BLAST analysis using Blast2GO (Conesa et al. 2005), searching the NCBI other genomic and other EST databases, and BLASTer (Thompson and Raub, 2012), which queried the UniProtKB database.  More than 1500 hits were found in both databases for both species.  Here we describe new polymorphic microsatellites and the identification of potential coding sequences in the unexplored butternut genome, along with our findings’ future applications in research on butternut and its relatives.