We are characterizing the molecular allelic diversity of S-RNases at the self-incompatibility (SI) locus in a natural population of Nicotiana alata. RT-PCR reactions were conducted on style RNA of eight plants, using degenerate oligonucleotides based on the conserved domains C1 and C5 of solanaceous S-RNases. Amplification products of expected size were cloned and sequenced, rendering two S-RNases previously characterized as SI alleles and three ribonucleases not reported yet as S-alleles, tentatively named D63, S5 and S7m. Genomic PCR with S5, S7m and D63 homologous primers revealed that the two first alleles were specific in six and one plants, respectively. By contrast, D63 was present in all plants examined, including a set of plants coming from a laboratory population. Spatial and temporal relative expression of S5 and S7m showed these alleles in developing and mature styles but not in floral tissues or leaves, paralleling the expression of well-known S-RNases. Conversely, expression of D63 ribonuclease was similar in different tissues and showed earlier expression respect to S5 and S7m in developing styles. Taken together these results strongly suggest that S5 and S7m are S-RNases and that D63 is a constitutive ribonuclease, not involved in SI. Genetic crosses and segregation analysis are in progress to demonstrate whether S5 and S7m are functional S-alleles.
