Radovan I. Bošković

Correlation of ribonuclease zymograms and incompatibility genotypes in almond

Proteins were extracted from styles of 29 self-incompatible cultivars of almond and separated using non-equilibrium pH gradient electro-focusing, and the gels were stained for ribonuclease activity. Mutually incompatible cultivars had similar banding patterns and, for the 24 cultivars already genotyped in France or California, the bands correlated well with the reported alleles. The band corresponding to S1 of the French labelling system was indistinguishable from that corresponding to Sb of the Californian labelling system, and a controlled cross confirmed that these alleles are identical. The band corresponding to the Californian Sa was distinct from the bands corresponding to French alleles and, to harmonise the allele labels, it was redesignated S5. The genotypes of five uncharacterised self-incompatible cultivars were inferred from zymograms as follows: ‘Desmayo Largueta’ and ‘Glorieta’, S1S5, ‘Masbovera’, S1S9, ‘Tarragones’, S2S9, and ‘Tokyo’, S6S7. The alleles designated S6 and S9 have not previously been reported. Nine self-compatible cultivars or selections were analysed, and each showed a band corresponding to an incompatibility allele as well as a common band; however, the correspondence of this common band to Sf, the allele for self-compatibility, is unproven.

Inheritance of stylar ribonucleases in cherry progenies, and reassignment of incompatibility alleles to two incompatibility groups

Stylar proteins were extracted from parents and seedlings of six progenies of cherry (Prunus avium), separated using isoelectric focusing, and the gels stained for ribonuclease activity. The zymogram of each plant showed two main ribonuclease bands in the region pI 8.3 to 9.6. Progenies from crosses of parents with one band in common segregated into just two classes, whereas progenies from crosses of parents with no common bands segregated into four classes, the two types of segregation corresponding to those expected from semi-compatible and fully-compatible crosses respectively. This behaviour was consistent either with the ribonuclease locus being tightly linked with the self-incompatibility, S, locus, or else with the S locus coding for the ribonuclease variants. Evidence favouring the latter hypothesis is discussed. An apparently anomalous segregation led us to assign to ‘Bradbourne Black’ a genotype different from that previously reported, and analysis of some other cultivars in the same incompatibility group, Group VII, led us to conclude the genotype of this group is S3S5, and not S4S5 as previously reported. Correspondingly, we suggest the genotype of Group V is S4S5, and not S3S5. Five new S alleles, S7, S8, S9, S10 and S11 were proposed in parental cultivars and selections that had not previously been assigned a genotype.

Morphological characteristics, microsatellite fingerprinting and determination of incompatibility genotypes of Sicilian sweet cherry cultivars

Summary Sicily has extensive germplasm of diploid sweet cherry (Prunus avium L.) that has not been well studied. In this investigation, 39 cherry accessions, selected from collections and farms, were analysed using molecular markers and characterised for various morphological and other agronomic characters such as flesh colour, fruit size, quality and, in some cases, ripening periods. Thirteen Simple Sequence Repeat (SSR) primer pairs, as well as two primer pairs for the incompatibility (S) locus, which amplified across the first intron of the S-RNase gene and across the intron of the SFB gene, were used in three multiplexed reactions to analyse the accessions. The number of alleles per SSR locus ranged from four to 11 (mean 7.2). Twelve S-alleles were found. Allele S4, which has been reported to be common in sweet cherry, was absent from the Sicilian germplasm and alleles S16 and S22 were frequent, although previously reported largely in wild populations. The accessions were assigned to their incompatibility groups. A UPGMA dendrogram was constructed and revealed possible synonyms. The set of SSR primers amplified unique fingerprints for 27 accessions, while twelve fell into six non-distinguishable groups. This approach could be used for comparing cultivars and wild accessions from other regions, and for establishing a database of this important species for breeding and conservation purposes. Several accessions useful for breeding are reported.

Characterisation of novel S-alleles from cherry (Prunus avium L.)

In plant populations exhibiting gametophytic self-incompatibility, individuals harbouring rare S alleles are likely to have a reproductive advantage over individuals having more common alleles. Consequently, determination of the self-incompatibility haplotype of individuals is essential for genetic studies and the development of informed management strategies. This study characterises six new S alleles identified in wild cherry (Prunus avium L.). Investigations to determine the S genotype of individuals in recently planted woodland through length polymorphisms of introns associated with the stylar S-RNase gene and the pollen SFB gene revealed six S intron profiles which did not correspond to those of known S alleles. These are now attributed to S27 to S32. Consensus primers, annealing in the S-RNase sequence coding for the signal peptide and C5 regions, were used to isolate the S-RNase alleles associated with the novel S intron profiles. The proteins corresponding to the new alleles were separated by isoelectric focusing from stylar extracts and their pI values determined. Similarities between the deduced amino acid sequence for the new alleles isolated and other cherry S-RNase sequences available on the databases ranged from 40% to 86%. Amplification products for SFB introns ranged from 172 to 208bp. New sequence regions exposed to positive selection were identified and the significance of the PS3 region reinforced. A phylogenetic relationship between P. avium S-RNases for S10 and S13 and between corresponding SFB alleles may indicate co-evolution of allele specificities of these two genes.

Genetic evidence that two independent S-loci control RNase-based self-incompatibility in diploid strawberry

The self-incompatibility mechanism that reduces inbreeding in many plants of the Rosaceae is attributed to a multi-allelic S locus which, in the Prunoideae and Maloideae subfamilies, comprises two complementary genes, a stylar-expressed S-RNase and a pollen-expressed SFB. To elucidate incompatibility in the subfamily Rosoideae, stylar-specific RNases and self-(in)compatibility status were analysed in various diploid strawberries, especially Fragaria nubicola and F. viridis, both self-incompatible, and F. vesca, self-compatible, and in various progenies derived from them. Unexpectedly, two unlinked RNase loci, S and T, were found, encoding peptides distinct from Prunoideae and Maloideae S-RNases; the presence of a single active allele at either is sufficient to confer self-incompatibility. By contrast, in diploid Maloideae and Prunoideae a single locus encodes S-RNases that share several conserved regions and two active alleles are required for self-incompatibility. Our evidence implicates the S locus in unilateral inter-specific incompatibility and shows that S and T RNases can, remarkably, confer not only allele-specific rejection of cognate pollen but also unspecific rejection of Sn Tn pollen, where n indicates a null allele, consistent with the the presence of the pollen component, SFB, activating the cognitive function of these RNases. Comparison of relevant linkage groups between Fragaria and Prunus suggests that Prunus S-RNases, unique in having two introns, may have resulted from gene conversion in an ancestor of Prunus. In addition, it is shown that there is a non-S locus that is essential for self-incompatibility in diploid Fragaria.