Cloning of six cherry self-incompatibility alleles and development of allele-specific PCR detection

Reverse transcription of stylar RNA from three cherry cultivars representing six self-incompatibility (S) alleles, Early Rivers (S1S2), Napoleon (S3S4) and Colney (S5S6), followed by 3 ' RACE using degenerate primers based on conserved regions of Prunus S ribonucleases (S RNases), gave six classes of partial putative S RNase clones. These were sequenced, and specific primers were designed for each class and, by using them in genomic PCR on 28 cultivars previously genotyped, we were able to assign the classes to individual S alleles. The primers for S-3 amplified the allele reported previously as S-8 and a controlled cross showed that these two alleles are functionally the same. Analysis of three cherry progenies using the specific primers showed cosegregation with stylar S RNases for all six clones. This confirmed that the clones indeed represent cherry S RNases. The allele-specific primers for S-3 presented here provide the first PCR test for true S-5. In a fourth progeny, the amplification product of a mutant S-4 allele, S-4', cosegregated with self-compatibility Sixteen cultivars were genotyped for the first time using the allele-specific primers. Thus, this approach will be valuable for genotyping cultivars and seedlings that have the alleles S-1-S-6 and for detecting self-compatible seedlings from vegetative material. The sequences of five of the S RNases, including S-5, were completed by 5 ' RACE.