Identification and cloning of related self-incompatibility S-genes in Papaver rhoeas and Papaver nudicaule

The primary goal of this study was to identify, clone and analyse new S-gene sequences in order to provide a basis for identifying amino acid residues that confer S-allele specificity. Three new putative S-alleles from Papaver rhoeas and Papaver nudicaule were identified using immunological and PCR methods. cDNAs encoding full-length open reading frames of the P. rhoeas S-8 and P. nudicaule Sn-1 genes were isolated. Nucleotide sequencing of these cDNAs, together with the partial S-7 sequence obtained by PCR, was used to derive the corresponding amino acid sequences. It is of interest that the P. nudicaule Sn-1 sequence, which is the first S-allele isolated from another species of Papaver, shares a closer sequence identity to the P. rhoeas S-3 amino acid sequence than S-3 does to S-1 from P. rhoeas. The identity of the S-8 allele was confirmed by expressing the coding region in Escherichia coli and demonstrating that the recombinant protein, designated S(8)e, specifically inhibited S-8 pollen in an in vitro bioassay. Information from sequence analysis of the S-8, Sn-1 and partial S-7 amino acid sequences revealed important information about Papaver S-proteins. It confirmed previous observations based on only two S-alleles, that whilst exhibiting a high degree of amino acid sequence polymorphism ranging from 51.3% to 63.7%, these molecules probably share very similar secondary structures. These studies also revealed that, in contrast to the S-proteins from the Solanaceae and Brassica, amino acid sequence variation is not found in hypervariable blocks, but instead, is found throughout the S-proteins, interspersed with numerous short strictly conserved segments.