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

Abstract

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.