The pollen coatings of both Brassica oleracea and Brassica napus contain a small family of basic 6-8 kDa proteins which are released on to the stigmatic surface on pollination. Following partial amino-acid sequencing of one of these pollen coat proteins (PCPs), PCR primers were constructed to isolate the PCP sequence from anther mRNA using RT-PCR. A cDNA was obtained which, in Northern hybridization experiments, revealed a characteristic pattern of expression during late stages of anther development. Interestingly, in situ hybridization revealed expression of this sequence to be confined to the cytoplasm of the trinucleate pollen grains: no signal was detected in the tapetum. Southern hybridization experiments have shown the gene (PCP1) to be a member of a large family of between 30 and 40 PCP genes in the genome of Brassica oleracea. Surprisingly, RFLP experiments showed reduced copy number (one to two copies) in some of the F2 segregants, perhaps resulting from the clustering of PCP sequences. PCP1 contains a single intron and encodes a small, basic peptide 83 amino acids in length featuring a hydrophobic signal peptide sequence separated from the more hydrophilic, cysteine-rich mature protein. The central part and C-terminal region of the peptide contain a characteristic and invariant pattern of eight cysteines which show clear homology with a number of other anther-specific genes; the remainder of the sequence shows little similarity to other sequences on the data bases. The product of PCP1 is a member of a large family of similar proteins, some of which have been demonstrated to bind specifically to S-locus glycoproteins, but does not appear to be genetically linked to the S-locus.

A new member of the S gene family, SLR3 (S-Locus Related 3), was identified in Brassica oleracea. This gene had a novel pattern of expression compared with previously described members of the family, being expressed in petals, sepals and vegetative apices, in addition to stigmas and anthers. Moreover, use of SLR3-derived probes in RNA blot and RACE-PCR (rapid amplification of cDNA ends-polymerase chain reaction) experiments has identified transcripts of genes closely related to SLR3 in leaves, cotyledons and, at high levels in developing anthers. SLR3 is not linked to the S locus but is linked to two or three closely related genes. Sequence analysis of the SLR3 gene indicates that it is derived from an ancestral receptor kinase gene that has been modified by a series of deletion events. As a result of these modifications, SLR3 is predicted to encode a secreted glycoprotein lacking both transmembrane and kinase domains. The putative SLR3 protein differs from the products of most other S gene family members in that several of the highly conserved cysteines have been lost. Within the S gene family, modification of receptor kinase genes by deletion may represent a general mechanism for the generation of genes encoding secreted glycoproteins.