Chromosome Walking in the Petunia Inflata Self-Incompatibility ( S -) Locus and Gene Identification in an 881-kb Contig Containing S 2 -RNase

Abstract

Self-incompatibility (SI) in the Solanaceae, Rosaceae and Scrophulariaceae is controlled by the polymorphic S locus, which contains two separate genes encoding pollen and pistil determinants in SI interactions. The S-RNase gene encodes the pistil determinant, whereas the pollen determinant gene, named the pollen S gene, has not yet been identified. Here, we set out to construct an integrated genetic and physical map of the S locus of Petunia inflata and identify any additional genes located at this locus. We first conducted chromosome walking at the S 2 locus using BAC clones that contained either S 2 -RNase or one of the nine markers tightly linked to the S locus. Ten separate contigs were constructed, which collectively spanned 4.4 Mb. To identify additional genes located at the S 2 locus, a 328-kb region (part of an 881-kb BAC contig) containing S 2 -RNase was completely sequenced. Approximately 76% of the region contained repetitive sequences, including transposon-like sequences. Other than S 2 -RNase , an F-box gene, named PiSLF 2 ( S 2 -allele of P. inflata S -locus F-box gene), was the only predicted gene whose deduced amino acid sequence was similar to the sequences of known proteins in the database. Two different cDNA selection methods were used to identify additional genes in the 881-kb contig; 11 groups of cDNA clones were identified in addition to those for S 2 -RNase and PiSLF 2 . RT-PCR analysis of expression profiles and PCR analysis of BAC clones and genomic DNA confirmed that seven of these 11 newly identified genes were located in the 881-kb contig.