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 PrunusS 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 S3 amplified the allele reported previously as S8, 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 S5 presented here provide the first PCR test for true S5. In a fourth progeny, the amplification product of a mutant S4 allele, S4′, 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 S1–S6 and for detecting self-compatible seedlings from vegetative material. The sequences of five of the S RNases, including S5, were completed by 5′ RACE.