John Carlson at Pennsylvania State University screened a subset of the above predicted SSRs.
Methods
DNA from twelve black cherry trees was collected for microsatellite screening from unrelated trees in a seed orchard at Penn Nursery (Pennsylvania Department of Conservation and Natural Resources, Bureau of Forestry, Spring Mills, PA), including parent trees R-14 and 11 of a genetic mapping population. DNA was extracted using a modified CTAB method, and DNA quality was assessed on 1% agarose gels. Species primer sets were tested against all collected genotypes within a species with a PCR reaction mixture containing between 4-6 ng DNA per reaction volume, and PCR cycles as follows: 95°C-5 min, 35 cycles of 95°C- 30s, 60°C-45s, 72°C-1 min, and a final 72°C-10 min. Both amplification and polymorphism were detected on 2% agarose gels stained with 0.05% (w/v) ethidium bromide. Polymorphism was estimated based on band width.
Results
Of the 96 tested SSRs, 48 successfully amplified and 26 were found to be polymorphic.
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