The work aimed to develop a reliable and convenient PCR approach for determining incompatibility S genotypes in almond. Initially, genomic DNAs of 24 accessions of known S genotype were amplified with novel consensus primers flanking the first and second introns of the S-RNase gene. The PCR products separated on agarose showed length polymorphisms and correlated well with the reference alleles S 1 -S 23 and S f . In addition, to improve discrimination between alleles of similar sizes, the same sets of primers but fluorescently labelled were used, and the products sized on an automated sequencer. These fluorescent primers were particularly informative in the case of the first intron, variation in the length of which has not been used previously for S genotyping in almond. Some reference alleles showed the same patterns with first and second intron primers, and others showed a microsatellite-like trace. Subsequently, the S genotypes of 26 cultivars not genotyped previously and of four of uncertain genotype were determined. An allele described in Australian work as putative S 10 was shown to be a ÔnewÕ allele and ascribed to S 24 and evidence of five more ÔnewÕ S alleles was found, for which the labels S 25 -S 29 are proposed. This PCR approach should be useful for genotyping in other Prunus crops.Most cultivars of almond [Prunus dulcis (Mill.) D.A. Webb] are self-incompatible and some are cross-incompatible (Tufts and Philp 1922). The incompatibility system is of the gametophytic type and controlled by a multi-allelic S locus (Gagnard 1954). To obtain good yields at least two cross-compatible cultivars of coincident flowering time should be planted together.Initial studies conducted to determine incompatibility genotypes in almond cultivars used controlled crosses. CrossaRaynaud and Grasselly (1985) proposed the existence of six different self-incompatibility alleles (S 1 , S 2 , S 3 , S 4 , S 7 and S 8 ) and the self-compatible allele S f in various European cultivars. Later, Kester et al. (1994) assigned four S alleles (S a , S b , S c and S d ) to explain the incompatibility groups of some American cultivars studied.Finding that almond incompatibility S alleles code for stylar proteins with ribonuclease activity that can be separated electrophoretically by isoelectric focusing (IEF) and non-equilibrium pH gradient electrofocusing (NEPHGE), Bosˇkovic´et al. (1997) determined the S genotype of 29 almond cultivars. This study corroborated the results obtained previously by Crossa-Raynaud andGrasselly (1985) andKester et al. (1994), and identified S 1 with S b , numbered S a as S 5 , and proposed two more alleles, S 6 and S 9 . In subsequent studies, the alleles S 10 (Bosˇkovic´et al. 1999), and S 11 and S 12 (Bosˇkovic´et al. 1998) were indicated and two more cultivars genotyped. Recently, 35 more cultivars (mostly of American origin) were genotyped with the same techniques and the alleles S 13

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Cross-compatibility relationships in almond are controlled by a gametophytically expressed incompatibility system partly mediated by stylar RNases, of which 29 have been reported. To resolve possible synonyms and to provide data for phylogenetic analysis, 21 almond S-RNase alleles were cloned and sequenced from SP (signal peptide region) or C1 (first conserved region) to C5, except for the S29 allele, which could be cloned only from SP to C1. Nineteen sequences (S4, S6, S11-S22, S25-S29)) were potentially new whereas S10 and S24 had previously been published but with different labels. The sequences for S16 and S17 were identical to that for S1, published previously; likewise, S15 was identical to S5. In addition, S4 and S20 were identical, as were S13 and S19. A revised version of the standard table of almond incompatibility genotypes is presented. Several alleles had AT or GA tandem repeats in their introns. Sequences of the 23 distinct newly cloned or already published alleles were aligned. Sliding windows analysis of Ka/Ks identified regions where positive selection may operate; in contrast to the Maloideae, most of the region from the beginning of C3 to the beginning of RC4 appeared not to be under positive selection. Phylogenetic analysis indicated four pairs of alleles had "bootstrap" support > 80%: S5/S10, S4/S8, S11/S24, and S3/S6. Various motifs up to 19 residues long occurred in at least two alleles, and their distributions were consistent with intragenic recombination, as were separate phylogenetic analyses of the 5' and 3' sections. Sequence comparison of phylogenetically related alleles indicated the significance of the region between RC4 and C5 in defining specificity.