Rapid identification of apple (Malus×domestica Borkh.) S alleles using sequencing-based DNA marker APPLid

All apple cultivars harbor the trait called self-incompatibility. Self-incompatibility represents that the pistils of the flowers are not successfully fertilized with own, the same cultivar’s pollens. Compatibility or incompatibility of apple flowers are determined by S alleles. For example, the most popular apple cultivar ‘Fuji’ possesses the S1 and S9 alleles (S1S9). Thus, ‘Fuji’ is incompatible with S1S9 cultivars, but is compatible with the cultivars possessing different combinations of S alleles such as S2S7 and S1S7. Apple S alleles have been identified by performing a series of allele-specific PCR amplifications, to detect more than ten different S alleles separately. Here, we developed a new type of sequencing-based DNA marker of the apple S-RNase gene, which identifies S alleles. This DNA marker was named APPLid (apple S-allele identifier). A 53-base region in the first coding sequence of S-RNase is the target of APPLid sequencing. Variation in nucleotide sequences in this APPLid sequence enables allele identifications. This region is amplified from apple genomic DNA by using a pair of degenerate primers. The forward primer is attached with ‘DS5 adaptor.’ After PCR amplification, electrophoresis and gel extraction of 177-bp DNA fragments, APPLid sequence is determined by direct sequencing with a sequencing primer. The APPLid sequences of 20 apple cultivars completely matched their S alleles, which include triploid cultivars. In conclusion, APPLid identifies apple S alleles (S1, S2, S3, S4, S5, S7, S9, S10, S20, S24, S25, S26, S27 and S28, so far) just by a single sequencing analysis