Primers complementary to the adaptor sequences plus one selective nucleotide (EcoRI + A and MseI + C) were used for preselective
amplification by PCR using nucleotide A, a selective amplification by PCR was performed with four combinations of EcoRI + 3 and MseI + 3 primers with the following PCR specifications: 94[degrees]C for 30 s; 20 cycles of 56[degrees]C for 60 s and 72[degrees]C for 60 s (pre-amplification); 94[degrees]C for 30 s; 6 cycles of 56[degrees]C for 30 s and 72[degrees]C for 60 s; 94[degrees]C for 30 s; and 23 cycles of 56[degrees]C for 30 s and 72[degrees]C for 60 s (selective amplifications).
After the preselective
amplification step, 26 primer combinations were selected for genotyping the parents and 80 progeny of the [F.
PCR, 2 [micro]L of diluted restriction/ligation product was added to 7.
5[micro]l dNTPs (5mM), 1[micro]l AFLP preselective
primer (EcoRI+ A-3'), 1 [micro]l AFLP preselective
primer (Tru9'+ C-3'), 0.
PCR amplification was performed using the Applied Biosystems AFLP kit (Applied Biosystems, Foster City, California).
amplification was performed with PstI+A and MseI+C primers and a PCR profile from Marques et al.
primers, selective primers, and PCR reagents were purchased from PE.
6 Thus, the general rule is: so long as the characteristics on which the discrimination/ranking is based are not preselective
in a way that violates equality and justice, they may be used as distinguishing parameters.
Following the preselective
amplification step, 26 primer combinations were selected for genotyping the parents and 80 progeny.
For the selective amplification, the reactions were set up as follows: 2[micro]l of 5-fold diluted preselective
amplification reaction product, 1.
Primer and adaptor oligonucleotide sequences used in the study Primer or adaptor oligonucelotide Sequence (5[right arrow]3') rDNA primers SR6R AAGTARAAGTCGTAACAAGG LR1 GGTTGGTTTCTTTTCCT M13 fingerprinting primer Phage M 13 GAGGGTGGCGGTTCT EcoRI adapters EA1 CTCGTAGACTGCGTACC EA2 CATCTGACGCATGGTTAA MseI adapters MA1 GACGATGAGTCCTGAG MA2 TACTCAGGACTCAT Preselective
primers EcoRI-T GACTGCGTACCAATTCT EcoRI-G GACTGCGTACCAATTCG MseI-C GATGAGTCCTGAGTAAC MseI-G GATGAGTCCTGAGTAAG Selective primers EcoRI-TG 6 FAM-GACTGCGTACCAATTCTG EcoRI-GT 6 FAM-GACTGCGTACCAATTCGT MseI-CA GATGAGTCCTGAGTAACA MseI-GT GATGAGTCCTGAGTAAGT Table 2.