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

Reverse transcription of stylar RNA from three cherry cultivars representin g 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 o f partial putative S RNase clones. These were sequenced, and specific prime rs were designed for each class and, by using them in genomic PCR on 28 cul tivars previously genotyped, we were able to assign the classes to individu al 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 functionall y the same. Analysis of three cherry progenies using the specific primers s howed cosegregation with stylar S RNases for all six clones. This confirmed that the clones indeed represent cherry S RNases. The allele-specific prim ers for S-3 presented here provide the first PCR test for true S-5. In a fo urth progeny, the amplification product of a mutant S-4 allele, S-4', coseg regated with self-compatibility Sixteen cultivars were genotyped for the fi rst time using the allele-specific primers. Thus, this approach will be val uable for genotyping cultivars and seedlings that have the alleles S-1-S-6 and for detecting self-compatible seedlings from vegetative material. The s equences of five of the S RNases, including S-5, were completed by 5 ' RACE .