Molecular and genetic characterization of the S locus in Hordeum bulbosum L., a wild self-incompatible species related to cultivated barley.

Gametophytic self-incompatibility (GSI) in the grasses is controlled by a distinct two-locus genetic system governed by the multiallelic loci S and Z. We have employed diploid Hordeum bulbosum as a model species for identifying the self-incompatibility (SI) genes and for elucidating the molecular mechanisms of the two-locus SI system in the grasses. In this study, we attempted to identify S haplotype-specific cDNAs expressed in pistils and anthers at the flowering stage in H. bulbosum, using the AFLP-based mRNA fingerprinting (AMF, also called cDNA-AFLP) technique. We used the AMF-derived DNA clones as markers for fine mapping of the S locus, and found that the locus resided in a chromosomal region displaying remarkable suppression of recombination, encompassing a large physical region. Furthermore, we identified three AMF-derived markers displaying complete linkage to the S locus, although they showed no significant homology with genes of known functions. Two of these markers showed expression patterns that were specific to the reproductive organs (pistil or anther), suggesting that they could be potential candidates for the S gene.

Visualization of the S-locus region in Ipomoea trifida: toward positional cloning of self-incompatibility genes.

Self-incompatibility (SI) in Ipomoea trifida is regulated by a single S locus with multiple alleles. Identification of SI genes in the S -locus region by positional cloning is one of the most important goals for understanding sexual reproduction in this species. Despite our intensive efforts to construct bacterial artificial chromosome (BAC) contigs covering the S -locus region, a gap was observed in the core region of the potential S locus. In order to confirm the physical linkage of two non-overlapping BAC contigs in the S -locus region and to determine the size of the gap between them, fluorescence in-situ hybridization (FISH) was performed on mitotic chromosomes and extended DNA fibres using previously isolated S -linked BAC clones as probes. The information obtained from this work would be useful for molecular cloning of the SI genes by a chromosome walking approach. In addition, we showed that strong suppression of recombination in the S locus was not related to the centromere because the S locus was mapped to one end of a chromosome.

Insights into the evolution of self-compatibility in Lycopersicon from a study of stylar factors.

To elucidate the molecular basis of loss of self-incompatibility in Lycopersicon, S-RNases and HT-proteins were analysed in seven self-compatible (SC) and three self-incompatible (SI) taxa. No or low stylar RNase activity was a common feature in most SC taxa examined, in contrast to the uniformly high levels of activity found in all SI species. The S-RNase gene is most likely deleted in the four red-fruited SC taxa (L. esculentum, L. esculentum var. cerasiforme, L. pimpinellifolium and L. cheesmanii) because S-RNase genes could not be amplified from genomic DNA. S-RNase genes could, however, be amplified from the genomes of the three green-fruited SC taxa examined. L. chmielewskii and L. hirsutum f. glabratum show a decreased accumulation of transcripts, possibly reflecting changes in the 5' flanking regions of the S-RNase genes. The remaining green-fruited SC species, L. parviflorum, has a functional S-RNase gene in its genome that is expressed at high levels in the style, suggesting a genetic factor responsible for the low S-RNase activity. Together these results argue for several independent mutations in the S-RNase gene over the course of Lycopersicon diversification, and that loss of S-RNase function is unlikely to the primary cause of the loss of self-incompatibility. We also examined the HT-B genes that play a role in self-incompatibility. HT-B transcripts were markedly reduced in the styles of all the SC taxa examined. A scenario is described where a mutation causing reduced transcription of HT-B in an ancestral SI species was central to the loss of self-incompatibility in Lycopersicon.

Cultivated tomato has defects in both S-RNase and HT genes required for stylar function of self-incompatibility.

Cultivated tomato (Lycopersicon esculentum), a self-compatible species, evolved from self-incompatible (SI) species in the genus Lycopersicon following a breakdown of the self-incompatibility system. In order to elucidate the molecular basis of this breakdown in L. esculentum, we first analysed the stylar proteins with an in-gel assay for ribonuclease activity and 2D-PAGE. No S-RNase protein or its activity was detected in the style of L. esculentum. We then introduced the S6-RNase gene from an SI relative, L. peruvianum, into L. esculentum. However, the styles of transgenic plants expressing S6-RNase at levels comparable to those found in the L. peruvianum style were unable to reject self-pollen and L. peruvianum pollen in an allele-specific manner. This indicated that defect in the S-RNase expression was not the sole reason for the loss of self-incompatibility in tomato. The asparagine-rich HT protein, originally identified from the style of Nicotiana alata, is the other stylar factor involved in self-incompatibility reaction. We cloned and sequenced two distinct genes encoding HT-A and HT-B proteins from L. peruvianum (LpHT-A and LpHT-B) and L. esculentum (LeHT-A and LeHT-B). A frame shift mutation in the coding sequence of LeHT-A and a stop codon in the ORF of LeHT-B were found, and no LeHT-B transcript was detected in the style of L. esculentum. The results suggest that the breakdown of self-incompatibility in cultivated tomato is associated with loss-of-function mutations in both S-RNase and HT genes.