Assay Method
DNA was extracted from young leaves as described by Davis et al. (1998) with the modifications of Lunde et al. (2000). For development of SSR primer pairs from ISSR amplicons, refer to the publication (Gurcan et al., 2010). To characterize the SSRs, forward primers of polymorphic loci were labeled fluorescently with 6-FAM, HEX, or NED and 50 genotypes were amplified under the same PCR conditions as in the initial screening. DNA was amplified by PCR in a 15 ?L mixture containing 12 pM each of forward and reverse primers, 1.6 ?L of 10x Biolase NH4 reaction buffer, 0.036 mM MgCl2, 0.027 M of each of dNTP, 0.25 units of Biolase DNA polymerase, and 3?25 ng of template DNA. The thermal cycler program was 94?C for 5 min; followed by 35 cycles of 94 ?C for 40 s, 30 s at the annealing temperature, 72 ?C for 40 s; and a final 7 min extension step at 72 ?C. The products were separated on 3% agarose gels to verify amplification and polymorphism. A mapping population of 144 seedlings (Mehlenbacher et al. 2006) was amplified at polymorphic SSR loci using fluorescently labeled primers and the same PCR conditions as for characterization. Up to four PCR products were diluted, pooled and the mixture was submitted to the Central Services Laboratory (CSL) of the Center for Genome Research and Biocomputing (CGRB) at OSU where fragments were separated and sized with an ABI 3100 capillary electrophoresis instrument.
Scoring Method
DNA fragment sizes were determined using GeneScan and Genotyper software.
Number of Observed Alleles
9