18:	A functional genomic approach to the search for the pollen S-gene involved in self-incompatibility.

Authors:	McCubbin, Andrew, G.(A)
Affiliations:	(A): Penn State University
Presenter:	Makaroff, Christopher A., makaroca@muohio.edu

One of the major goals in self-incompatibility (SI) research is the identification of the pollen S-gene whose allelic products interact with the allelic products of the pistil S-RNasegene, S-RNases, to elicit SI responses. The pollen S-gene must fulfill two important criteria: (1) it must be physically linked to the S-RNase gene of the same S-haplotype, and (2) there must be sufficient sequence polymorphism between different pollen S-alleles in order for each to encode a unique allelic specificity. Through the use of differential display and subtractive hybridization, we have isolated 14 distinct pollen-expressed genes of Petunia inflata that all lie within a centimorgan of the S-RNase gene of the S-locus (i.e., no recombination was found from the analysis of at least 100 F2 plants segregating for S-alleles). None of these genes appear to be good candidates for the pollen S-gene, because the degree of their allelic sequence diversity is less than expected. However, they will serve as useful molecular markers in our undertaking a 'functional genomic' approach to search for the pollen S-gene. Having optimized the purification of megabase-sized DNA from P. inflata, we are in the process of using pulse-field gel electrophoresis to determine the physical distance between each of these S-locus markers and the S-RNase gene, and between pairs of these markers. Furthermore, we have constructed a bacterial artificial chromosome (BAC) library in the binary vector BIBAC2. BAC clones containing large fragments of the S-locus will be isolated and screened for the presence of the pollen S-gene using a plant transformation based functional assay.