Abstract
Despite being perfect hermaphrodites, most angiosperms have developed self-incompatibility as an important genetic mechanism to avoid self-fertilization and inbreeding, and to promote allogamy and outcrossing. The molecular mechanisms of gametophytic self-incompatibility (GSI) in the Rosaceae have been studied intensively, since fertilization represents an important agronomic parameter for stable fruit yield and breeding activities. In apple, GSI is controlled by an S locus with a number of S alleles. In the case of identical S alleles between male pollen and female stigma during pollination, stigmatic S-RNases are able to arrest growth of the pollen tube, preventing it from reaching and fertilizing the ovule. Knowledge of compatible cultivars is a basic requirement for the selection of appropriate pollen donors in production orchards as well as in breeding programs. Thus, to identify the S alleles of a cultivar, a series of different protocols have been developed using either allele-specific primer amplifications or allele-specific restriction enzymes. Both require individual assays for the detection of each single allele out of over 30, and thus lack efficiency in terms of time and money. Recently, a high-throughput method for genotyping S alleles via capillary electrophoresis has been developed that allows up to 49 different S alleles to be distinguished through the use of general amplification primers combined with two enzymatic restrictions. In the present study, the method has been optimized and applied to a set of 180 apple cultivars ranging from local cultivars to new commercial cultivars and breeding lines. With this protocol, all known S alleles of Malus x domestica could be differentiated in a single multiplexed run of capillary electrophoresis. Comparing the results to phenotypic fertilization tests allows the compatibility of cultivars to be predicted in future designs of apple orchards, as well as in future breeding activities.