Evaluation Method
DNA Extraction
DNA was extracted from young leaves with the PureGene kit (Gentra).
PCR Protocol
PCR reactions were performed in 10 uL volume containing 1X reaction buffer, 2 mM MgCl2, 0.2 mM dNTPs, 0.15 uM of each primer (if separation platform consisted of CEQ 8000) or 0.3 uM of each primer (if separation platform consisted of ABI 3100), 0.25 units of Biolase Taq DNA polymerase (Bioline Inc.), and 2.5 ng genomic DNA.
After initial denaturation at 94 C for 3 minutes, DNA was amplified for 35 cycles in an Eppendorf Gradient thermocycler or an MJ Research Tetrad thermocycler programmed for a 40 s denaturation step at 94 C, a 40 s at optimum annealing temperature, and a 40 s extension step at 72 C. A final extension step of 72 C for 30 minutes followed.
View a table of the SSR data as an Excel Spreadsheet.
Assay Method
DNA was extracted from young leaves with the PureGene kit (Gentra). PCR Protocol: PCR reactions were performed in 10 uL volume containing 1X reaction buffer, 2 mM MgCl2, 0.3 uM of each primer, 0.25 units of Biolase Taq DNA polymerase (Bioline Inc.), and 2.5 ng genomic DNA. After initial denaturation at 94 C for 3 min, DNA was amplified for 35 cycles in an Eppendorf Gradient thermocycler or an MJ Research Tetrad thermocycler programmed for a 40 s denaturation step at 94 C, a 40 s at optimum annealing temperature, and a 40 s extension step at 72 C. A final extension step of 72 C for 30 min followed.
Scoring Method
1 ul of a mix of three or four PCR products was separated on an ABI 3100 capillary electrophoresis instrument (Applied Biosystems, Foster City, California) at the Central Services Laboratory (CSL) of the Center for Genome Research and Biocomputing (CGRB) at Oregon State University (OSU). DNA fragment sizes were determined using GeneScan and Genotyper software.
Number of Observed Alleles
3
Size of Alleles
155-164 bp