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-incompatibility mechanism that enables the pistil to reject self pollen but accept non-self pollen for fertilization. The pistil function in this system has been 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-gene. As a first step in using a functional genomic approach to identify the pollen S-gene, a genomic BAC (bacterial artificial chromosome) library of the S2S2 genotype of Petunia inflata, a self-incompatible solanaceous species, 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 genome coverage. Screening of the library using cDNAs for the S2-RNase gene and 13 pollen-expressed genes that are linked to the S-locus yielded 51 positive clones, with at least one positive clone for each gene. Collectively, at least 2 Mb of the chromosomal region was spanned by these clones. Together, three clones that contained the S2-RNase gene spanned approximately 263 kb. 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 discussed.
McCubbin
A G
AG
Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park 16802, USA.
Zuniga
C
C
Kao
T
T
D022202:Chromosomes, Artificial, Bacterial; D003001:Cloning, Molecular / Q000379:methods; D018506:Gene Expression Regulation, Plant; D015723:Gene Library; D017343:Genes, Plant; D018745:Genome, Plant; D010446:Peptide Fragments / Q000235:genetics*; D011058:Pollen / Q000235:genetics*; D012260:Ribonucleases / Q000235:genetics*; D019657:Solanaceae / Q000235:genetics*
D010446:Peptide Fragments; C023560:ribonuclease S-peptide; D012260:Ribonucleases