As part of the almond breeding programme at IRTA, we investigated the S genotypes of several cultivars using a combination of RNase zymograms, testcrosses, pollen-tube growth analysis and molecular identification by PCR analysis. For some of the cultivars examined, discrepancies appeared between their S alleles as reported in the literature and those found in this investigation, leading to a re-evaluation of their S genotypes. Analysis of the stylar ribonucleases (RNases), which are known to correlate with S alleles, of cvs. Achaak, Ardechoise, Desmayo Largueta, Ferrastar, Gabaix, Garbi, Glorieta, Languedoc, Primorskiy and Texas revealed inconsistencies with respect to the S5 and S10 alleles. However, PCR with the conserved primer pair AS1II/AmyC5R failed to detect any of these inconsistencies. When the S alleles from Desmayo Largueta, Gabaix, Primorskiy and Texas were sequenced, Texas and Primorskiy were found to carry the reported S5 allele, while Desmayo Largueta and Gabaix carried a new allele, which has been tentatively denoted as S25 This new S allele, previously reported to be S10, was also identified in Achaak, Ardechoise and Ferrastar. The proposed new S genotypes are Achaak (S2S25), Ardechoise (S1S25), Desmayo Largueta (S1S25), Ferrastar (S2S25) and Gabaix (S10S25). The S alleles of Garbi, Glorieta, Languedoc, Texas and Primorskiy remain as reported in the literature. Testcrosses in the field and laboratory confirmed the new S genotypes. One cultivar (Gabaix) could be assigned to the existing cross-incompatibility group O of unique genotypes, and two new groups were established (XVI and XVII) consisting of two cultivars each. The clarification of these S alleles will be useful in almond breeding programmes and for planning new commercial orchards in the future.

To compile self-(in)compatibility almond genotypes, a review of 133 commercial cultivars of wide geographical origin was made. The information gathered from own and mainly published work will be useful for both grower's cultivar choice when planting and for breeder's cross design when planning. The almond S genotypes compiled were identified using five different methods: biological (pollination tests in the field and in the laboratory) and molecular (RNases, PCR and sequencing). In most cases, genotypes were assigned after combining more than one technique. Cultivars were classified into three categories: self-incompatible (99), self-compatible (16) and doubtful self-incompatible (18). The database is divided in 9 fields (name, origin, parentage, obtention year (crossing, selection or release), S genotype, technique used, reference, consensus genotype, and cross incompatibility group). A study of the 27 S alleles already identified and their geographical distribution within the cultivated almond is also presented. The study was divided into cultivars of known and unknown parentage and the distribution of S alleles frequencies was uneven among the 133 cultivars. S allele frequencies are related to geographical origin. Some alleles (S 1 , S 5 , S 7 and S 8 ) are more frequently observed than the others among cultivars of both known and unknown parentage. In the cultivated almond, the S f allele is only found in the Puglia region, Italy. The S f frequency is three times higher in cultivars released from breeding programmes than in cultivars selected by growers. From the 351 resulting possible genotypes by combination of the 27 S alleles identified only 20 CIG (0-XIX) have been established, which represents a small fraction of the whole genetic diversity of this polymorphic gene in almond.

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To verify the compatibility behaviour of the almond cultivar 'Francolı´' and to clarify its S genotype a combination of pollination tests, stylar ribonuclease and allele specific PCR analysis was used. 'Francolı´' was released from IRTA's breeding programme in 1994, having been putatively raised from the cross 'Cristomorto' (S 1 S 2 ) · 'Gabaix' (S 10 S 25 ). This cultivar was also reported to be self-incompatible but revealing only one S band in the zymograms after S-RNases analysis. 'Francolı´' sets nuts after test crossing with two S 1 S 25 cultivars, having a different genotype from that earlier reported. 'Francolı´' was also observed to be self-compatible after selfing flowers in the field and in the laboratory. 'Francolı´' was re-assigned the S 1 S f genotype after test crossing, stylar ribonuclease and PCR data analysis. After microsatellite analysis, the self-compatible 'Tuono' (S 1 S f ) cultivar is suggested as the male parent of 'Francolı´' instead of the earlier reported 'Gabaix'.

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