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

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 alleles. For example, the most popular apple cultivar 'Fuji' possesses the and alleles ( ). Thus, 'Fuji' is incompatible with cultivars, but is compatible with the cultivars possessing different combinations of alleles such as and . Apple alleles have been identified by performing a series of allele-specific PCR amplifications, to detect more than ten different alleles separately. Here, we developed a new type of sequencing-based DNA marker of the apple gene, which identifies alleles. This DNA marker was named (apple -allele identifier). A 53-base region in the first coding sequence of is the target of sequencing. Variation in nucleotide sequences in this 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, sequence is determined by direct sequencing with a sequencing primer. The sequences of 20 apple cultivars completely matched their alleles, which include triploid cultivars. In conclusion, identifies apple alleles ( , , , , , , , , , , , , and , so far) just by a single sequencing analysis.