Characterization of the S-locus controlling self-incompatibility in Petunia inflata

Self-incompatibility (SI) is a genetic barrier to inbreeding in flowering plants. In Petunia inflata, SI is controlled by the multiallelic S-locus. Interactions between the S-allele products in pollen and in pistil result in rejection of self-pollen and acceptance of non-self pollen by the pistil. The gene that controls the pistil function in SI interactions has been identified and is named the S-RNase gene, because its allelic products have RNase activity. Several lines of evidence suggest that the S-RNase gene does not control the pollen function in SI. Thus, the S-locus must contain another polymorphic gene, named the pollen S-gene, whose allelic products interact with S-RNases in an allele-specific manner to elicit SI responses.; Several approaches were used to identify the pollen S-gene. First, yeast two-hybrid protein-protein interaction assay was used to identify pollen proteins that interact with the pistil S-RNase (Chapter 2). None of the five positive clones identified in the assay turned out to encode true S-RNase interacting proteins upon further analysis. Second, mRNA differential display was used to identify pollen cDNAs that were amplified in an S-allele-specific manner (Chapter 3). Twelve pollen-expressed genes that are tightly linked to the S-RNase gene were identified. Although none of them corresponded to the pollen S-gene judging from their low levels of allelic sequence diversity, they served as DNA markers for the map-based cloning approach, which was the third approach used to identify the pollen S-gene (Chapter 4). To determine the genetic and physical distance between the 12 cDNA markers and S-RNase, recombinational analysis and pulsed-field gel electrophoresis were performed. The results suggest that four of the markers were 0.1 and 0.2 cM away from the S-RNase, the other eight markers and the S-RNase gene are located in a large chromosomal region (>1 mb) where recombination occurs very infrequently. A BAC (Bacterial Artificial Chromosome) contig spanning an approximately 328 kb region containing the S2-RNase gene was identified, and direct cDNA selection and RNA blot analysis were used to identify expressed genes in this S -locus region. After the availability of the sequence of the region, several computation-based methods were used to identify potential genes. Among the 10 most promising putative genes identified, the most interesting one is a 1.4 kb intronless gene (named 251k), which is 160 kb from the S 2-RNase. Its deduced amino acid sequence is highly similar to that of an S-locus F-box (SLF)-S2 protein (GeneBank accession no: AJ297974). It was further characterized.; The S-locus is one of the most polymorphic loci known in plants, but little is known about the mechanism of the generation of its allelic diversity. RT-PCR was used to obtain cDNAs for 16 alleles of the S-RNase gene that had been identified in a natural population of Petunia inflata . Sequence analyses of these cDNAs showed strong evidence of intragenic recombination between certain allele pairs of the S-RNase gene, suggesting that intragenic recombination is likely to have contributed to the allelic diversity at the S-locus (Chapter 5).