Construction of two BAC libraries from the wild Mexican diploid potato, Solanum pinnatisectum, and the identification of clones near the late blight and Colorado potato beetle resistance loci

Abstract

To facilitate isolation and characterization of disease and insect resistance genes important to potato, two bacterial artificial chromosome (BAC) libraries were constructed from genomic DNA of the Mexican wild diploid species, Solanum pinnatisectum, which carries high levels of resistance to the most important potato pathogen and pest, the late blight and the Colorado potato beetle (CPB). One of the libraries was constructed from the DNA, partially digested with BamHI, and it consists of 40328 clones with an average insert size of 125 kb. The other library was constructed from the DNA partially digested with EcoRI, and it consists of 17280 clones with an average insert size of 135 kb. The two libraries, together, represent approximately six equivalents of the wild potato haploid genome. Both libraries were evaluated for contamination with organellar DNA sequences and were shown to have a very low percentage (0.65-0.91%) of clones derived from the chloroplast genome. High-density filters, prepared from the two libraries, were screened with ten restriction fragment length polymorphism (RFLP) markers linked to the resistance genes for late blight, CPB, Verticillium wilt and potato cyst nematodes, and the gene Sr1 for the self-incompatibility S-locus. Thirty nine positive clones were identified and at least two positive BAC clones were detected for each RFLP marker. Four markers that are linked to the late blight resistance gene Rpi1 hybridized to 14 BAC clones. Fifteen BAC clones were shown to harbor the PPO (polyphenol oxidase) locus for the CPB resistance by three RFLP probes. Two RFLP markers detected five BAC clones that were linked to the Sr1 gene for self-incompatibility. These results agree with the library's predicted extent of coverage of the potato genome, and indicated that the libraries are useful resources for the molecular isolation of disease and insect resistance genes, as well as other economically important genes in the wild potato species. The development of the two potato BAC libraries provides a starting point, and landmarks for BAC contig construction and chromosome walking towards the map-based cloning of agronomically important target genes in the species.