Construction of a binary bacterial artificial chromosome library of Petunia inflata and the isolation of large genomic fragments linked to the self-incompatibility (S-) locus

The Solanaceae family of flowering plants possesses a type of self-incompat ibility mechanism that enables the pistil to reject self pollen but accept non-self pollen for fertilization. The pistil function in this system has b een shown to be controlled by a polymorphic gene at the S-locus, termed the S-RNase gene. The pollen function is believed to be controlled by another as yet unidentified polymorphic gene at the S-locus, termed the pollen S-ge ne. As a first step in using a functional genomic approach to identify the pollen S-gene, a genomic BAC (bacterial artificial chromosome) library of t he S2S2 genotype of Petunia inflata, a self-incompatible solanaceous specie s, was constructed using a Ti-plasmid based BAC vector, BIBAC2. The average insert size was 136.4 kb and the entire library represented a 7.5-fold gen ome coverage. Screening of the library using cDNAs for the S-2-RNase gene a nd 13 pollen-expressed genes that are linked to the S-locus yielded 51 posi tive clones, with at least one positive clone for each gene. Collectively, at least 2 Mb of the chromosomal region was spanned by these clones. Togeth er, three clones that contained the S-2-RNase gene spanned similar to 263 k b. How this BAC library and the clones identified could be used to identify the pollen S-gene and to study other aspects of self-incompatibility is di scussed.