|Rinehart, Timothy - Tim|
Submitted to: HortScience
Publication Type: Abstract Only
Publication Acceptance Date: 6/1/2007
Publication Date: 7/1/2007
Citation: Wadl, P., Wang, X., Rinehart, T.A., Trigiano, R., Windham, M. 2007. Development of Microsatellite (SSR) Markers from Kousa Dogwood. HortScience. pp.919-1022.
Technical Abstract: Microsatellites or simple sequence repeats (SSRs) are stretches of DNA that consist of tandemly repeated mono-, di-, tri-, tetra- or penta-nucleotide units and occur in abundance in the genomes of most eukaryotes. SSR markers are preferred markers in plant breeding because of the uniform genome coverage, high levels of polymorphism, co-dominance and reproducibility. Microsatellite (SSR) markers for kousa dogwood, Cornus kousa Hance, were isolated using a biotin enrichment protocol. The method included ligation of 300–1000 base pair enzyme-digested fragments and adaptors, affinity capture of microsatellite repeat using biotinylated oligoprobes attached to streptavidin beads. A (GT)12 enriched microsatellite library was constructed from C. kousa genomic DNA. To isolate the SSR-containing clones, colony PCR of 288 clones with three primers (two vector primers and one primer corresponding to the (GT)12 repeat oligo) was performed. Sixty-one percent (176/288) of the screened colonies produced PCR products that exhibited a smear pattern in 2% metaphor agarose gels indicating SSR-containing clones. From the 176 potential SSR-containing clones, 48 clones were sequenced and 46 clones (96%) contained a SSR motif, but only six of the sequenced clones contained sufficient flanking regions to allow for primer design. For polymorphism assessment, six primer sets were tested using 32 kousa genotypes: one of the sets provided monomorphic products, whereas, two of the primer sets revealed polymorphic PCR products.