Although various consensus polymerase chain reaction (PCR) primers have been reported for identifying Prunus S-alleles, they have been developed from and optimized on a limited set of alleles, which may limit their applicability to a broader allele range. To develop a primer set for use across the genus, degenerate consensus primers were designed from conserved regions of 27 S-RNase sequences available from five Prunus species. The primers were tested in 15 previously genotyped cultivars of cherry, almond and apricot, representing alleles S 1 to S 6 in each crop and also S c in apricot. Comparisons were made with previously published primers tested in the same 15 cultivars under reported reaction conditions. The new primers generated an amplification product for each of the 19 S-alleles whereas those previously available amplified no more than 14. The primers will be useful for genotyping and genetic studies in cultivars and wild populations.

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

No abstract

Prunus dulcis, the almond, is a predominantly self-incompatible (SI) species with a gametophytic self-incompatibility system mediated by S-RNases. The economically important allele Sf, which results in self-compatibility in P. dulcis, is said to have arisen by introgression from Prunus webbii in the Italian region of Apulia. We investigated the range of self-(in)compatibility alleles in Apulian material of the two species. About 23 cultivars of P. dulcis (14 self-compatible (SC) and nine SI) and 33 accessions of P. webbii (16 SC, two SI and 15 initially of unknown status), all from Apulia, were analysed using PCR of genomic DNA to amplify S-RNase alleles and, in most cases, IEF and staining of stylar protein extracts to detect S-RNase activity. Some amplification products were cloned and sequenced. The allele Sf was present in nearly all the SC cultivars of P. dulcis but, surprisingly, was absent from nearly all SC accessions of P. webbii. And of particular interest was the presence in many SI cultivars of P. dulcis of a new active allele, labelled S30, the sequence of which showed it to be the wild-type of Sf so that Sf can be regarded as a stylar part mutant S30 degrees . These findings indicate Sf may have arisen within P. dulcis, by mutation. One SC cultivar of P. dulcis, 'Patalina', had a new self-compatibility allele lacking RNase activity, Sn5, which could be useful in breeding programmes. In the accessions of P. webbii, some of which were known to be SC, three new alleles were found which lacked RNase activity but had normal DNA sequences.

Current forestry policy promotes the use of local seed for new plantings, on the assumption that local material may be better adapted to local conditions. However, landscape-scale genetic studies which are necessary to underpin conservation and breeding strategies are often lacking. We investigated molecular diversity in common ash (Fraxinus excelsior L.) sampled from 42 British and six French sites with microsatellites. Chloroplast haplotype H04 was the most common and widespread in Britain, although rare and localized individuals with H02 and H09 were also detected. In addition, three new chloroplast haplotypes were identified, and these were rare and highly localized. In terms of nuclear microsatellite markers, allelic richness differed between sites and decreased in an east to west direction. Differentiation between sites was often very low (mean F(ST) 0.025), indicating few differences between the majority of sites. There was a clear excess of homozygotes (mean H(O) 0.669, mean H(E) 0.818) and a relatively high F(IS) (mean 0.182), suggests a consistent level of inbreeding or a widespread Wahlund effect in many F. excelsior sites. Gene pool ancestry analysis suggested that the majority of British F. excelsior belongs to a single meta-population which covers mainland western and central Europe. Three northern and western sites diverged markedly from the dominant population, and may represent remnants of two late potential Ice Age refugia in northern Britain. The data provide new information which will aid development of appropriate conservation policies for ash and other wind pollinated tree species.

The stylar products of the S-locus for the gametophytic self-incompatibility (GSI) system in the Rosaceae are ribonucleases (S-RNases). Recently, sequences for 13 pear S-RNase alleles have been published and named following a letter-symbol nomenclature (S a to S d and S h to S p ). To establish the correspondence between these sequences and the self-incompatibility alleles we have described previously (S 1 to S 5 ), we have amplified genomic DNA with consensus primers from the cultivars, ÔWilliamsÕ (S 1 S 2 ), ÔCosciaÕ (S 3 S 4 ), ÔButirra Precoce MorettiniÕ (S 1 S 3 ), ÔSanta Maria MorettiniÕ (S 2 S 3 ) and ÔDoyenne du ComiceÕ (S 4 S 5 ) and identified PCR products specifically associated with each S allele. Cloning and sequencing of the amplification products has revealed that they correspond to European pear sequences already deposited in the database. This allowed us to link S-RNase sequences with S allele phenotypes and to determine a correspondence between the symbol-letter nomenclature used to name S-RNase sequences and the number-based nomenclature used to name S alleles. Based on this result the prediction of new cross-incompatibilities among pear cultivars is discussed. Finally, we propose a unified number-based nomenclature to avoid future confusion denominating S alleles in pear.

Self-incompatibility in the genus Prunus is controlled by two genes at the S-locus, S-RNase and SFB. Both genes exhibit the high polymorphism and high sequence diversity characteristic of plant self-incompatibility systems. Deduced polypeptide sequences of three myrobalan and three domestic plum S-RNases showed over 97% identity with S-RNases from other Prunus species, including almond, sweet cherry, Japanese apricot and Japanese plum. The second intron, which is generally highly polymorphic between alleles was also remarkably well conserved within these S-allele pairs. Degenerate consensus primers were developed and used to amplify and sequence the co-adapted polymorphic SFB alleles. Sequence comparisons also indicated high degrees of polypeptide sequence identity between three myrobalan and the three domestic plum SFB alleles and the corresponding Prunus SFB alleles. We discuss these trans-specific allele identities in terms of S-allele function, evolution of new allele specificities and Prunus taxonomy and speciation.

Physocarpus opulifolius is a deciduous shrub native to North America belonging to the Spiraeoideae subfamily of the Rosaceae. The cultivars ÔLuteusÕ and ÔDiaboloÕ are grown in gardens for their ornamental foliage, golden and purple respectively. We developed a linkage map of P. opulifolius with a view to detecting markers for the leaf colour genes, which are under major gene control. A total of 162 molecular markers (128 RAPDs, 27 AFLPs, three RGA, three STS markers and one SSR) and the leaf colour genes Pur and Aur were scored in the Physocarpus progeny and used to create a linkage map covering 586.1 cM over nine linkage groups. There was an average of 18.2 markers per linkage group and a mean linkage group length of 65.1 cM. Both leaf colour genes were mapped. This is the first reported linkage map of a member of the Spireaeoideae and the mapping of a small number of transferable markers has demonstrated its utility to comparative mapping, which will complement existing comparative mapping efforts in other rosaceous subfamilies.