Allelic diversity of S-RNase alleles in diploid potato species.

KEY MESSAGE: The S-ribonuclease sequences of 16 S-alleles derived from diploid types of Solanum are presented. A phylogenetic analysis and partial phenotypic analysis support the conclusion that these are functional S-alleles. S-Ribonucleases (S-RNases) control the pistil specificity of the self-incompatibility (SI) response in the genus Solanum and several other members of the Solanaceae. The nucleotide sequences of S-RNases corresponding to a large number of S-alleles or S-haplotypes have been characterised. However, surprisingly, few S-RNase sequences are available for potato species. The identification of new S-alleles in diploid potato species is desirable as these stocks are important sources of traits such as biotic and abiotic resistance. S-RNase sequences are reported here from three distinct diploid types of potato: cultivated Solanum tuberosum Group Phureja, S. tuberosum Group Stenotomum, and the wild species Solanum okadae. Partial S-RNase sequences were obtained from pistil RNA by RT-PCR or 3'RACE (Rapid Amplification of cDNA Ends) using a degenerate primer. Full-length sequences were obtained for two alleles by 5'RACE. Database searches with these sequences identified 16 S-RNases in total, all of which are novel. The sequence analysis revealed all the expected features of functional S-RNases. Phylogenetic analysis with selected published S-RNase and S-like-RNase sequences from the Solanaceae revealed extensive trans-generic evolution of the S-RNases and a clear distinction from S-like-RNases. Pollination tests were used to confirm the self-incompatibility status and cross-compatibility relationships of the S. okadae accessions. All the S. okadae accessions were found to be self-incompatible as expected with crosses amongst them exhibiting both cross-compatibility and semi-compatibility consistent with the S-genotypes determined from the S-RNase sequence data. The progeny analysis of four semi-compatible crosses examined by allele-specific PCR provided further confirmation that these are functional S-RNases.

Trans-specific S-RNase and SFB alleles in Prunus self-incompatibility haplotypes.

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.

Loss of pollen-S function in two self-compatible selections of Prunus avium is associated with deletion/mutation of an S haplotype-specific F-box gene.

Recently, an S haplotype-specific F-box (SFB) gene has been proposed as a candidate for the pollen-S specificity gene of RNase-mediated gametophytic self-incompatibility in Prunus (Rosaceae). We have examined two pollen-part mutant haplotypes of sweet cherry (Prunus avium). Both were found to retain the S-RNase, which determines stylar specificity, but one (S3' in JI 2434) has a deletion including the haplotype-specific SFB gene, and the other (S4' in JI 2420) has a frame-shift mutation of the haplotype-specific SFB gene, causing amino acid substitutions and premature termination of the protein. The loss or significant alteration of this highly polymorphic gene and the concomitant loss of pollen self-incompatibility function provides compelling evidence that the SFB gene encodes the pollen specificity component of self-incompatibility in Prunus. These loss-of-function mutations are inconsistent with SFB being the inactivator of non-self S-RNases and indicate the presence of a general inactivation mechanism, with SFB conferring specificity by protecting self S-RNases from inactivation.

The F-box protein AhSLF-S2 controls the pollen function of S-RNase-based self-incompatibility.

Recently, we have provided evidence that the polymorphic self-incompatibility (S) locus-encoded F-box (SLF) protein AhSLF-S(2) plays a role in mediating a selective S-RNase destruction during the self-incompatible response in Antirrhinum hispanicum. To investigate its role further, we first transformed a transformation-competent artificial chromosome clone (TAC26) containing both AhSLF-S(2) and AhS(2)-RNase into a self-incompatible (SI) line of Petunia hybrida. Molecular analyses showed that both genes are correctly expressed in pollen and pistil in four independent transgenic lines of petunia. Pollination tests indicated that all four lines became self-compatible because of the specific loss of the pollen function of SI. This alteration was transmitted stably into the T1 progeny. We then transformed AhSLF-S(2) cDNA under the control of a tomato (Lycopersicon esculentum) pollen-specific promoter LAT52 into the self-incompatible petunia line. Molecular studies revealed that AhSLF-S(2) is specifically expressed in pollen of five independent transgenic plants. Pollination tests showed that they also had lost the pollen function of SI. Importantly, expression of endogenous SLF or SLF-like genes was not altered in these transgenic plants. These results phenocopy a well-known phenomenon called competitive interaction whereby the presence of two different pollen S alleles within pollen leads to the breakdown of the pollen function of SI in several solanaceaous species. Furthermore, we demonstrated that AhSLF-S(2) physically interacts with PhS(3)-RNase from the P. hybrida line used for transformation. Together with the recent demonstration of PiSLF as the pollen determinant in P. inflata, these results provide direct evidence that the polymorphic SLF including AhSLF-S(2) controls the pollen function of S-RNase-based self-incompatibility.