Identification of S-alleles in almond using multiplex PCR

The S-genotypes of eight almond (Prunus dulcis Miller (D.A. Webb)) cultivars from different geographical origins and of nine new selections from the CEBAS-CSIC (Murcia, Spain) breeding program were determined using single and multiplex PCR with different sets of specific oligonucleotide primers. The results of PCR using the AS1II- and AmyC5R-specific primers showed amplification in a single reaction of 10 different self-incompatibility alleles and of the self-compatibility allele Sf. However, the amplified fragments of the Sf allele were of similar sizes to those amplified from the S3 self-incompatibility allele. For this reason, a specific PCR primer CEBASf was designed from the intron sequence of Sf. A multiplex-PCR reaction using the AS1II, CEBASf and AmyC5R primers permitted unequivocal identification of the 10 self-incompatibility alleles and of the self-compatibility allele. Multiplex PCR opens the possibility to identify new S-alleles using different sets of primers. The applications of these PCR markers in the almond-breeding programs are discussed.