Long Range PCR: Promising Tool for Allelic Mining in Wild Potato Species

Sanchez, M.J. and J.M. Bradeen

Plant & Animal Genome XII (San Diego, CA Jan 2004)

The PCR (polymerase chain reaction) technique revolutionized molecular biology by the late 1980’s. Since then, distinct variations in the original procedure have been developed. Long range PCR (LR-PCR), a technique that amplifies long DNA fragments, is a promising gene-cloning tool. The RB gene, a functional late blight resistance gene, was isolated from the wild potato species Solanum bulbocastanum using LR-PCR (Song et al.; PNAS 100: 9128). The RB gene confers broad-spectrum resistance to several races of Phytophthora infestans under field conditions (Toluca, Mexico), which is highly desirable for potato production. Due to the high level of sequence homology among Solanum species, other functional RB alleles may potentially be cloned through the application of LR-PCR in different populations of Solanum bulbocastanum and other Solanum species. However, technical optimization is required for large-scale application across numerous genotypes. Two factors are crucial for LR-PCR performance: template DNA quality and amplification accuracy. DNA purity and integrity were tested for five commercial genomic DNA extraction kits. Likewise, amplification efficiency and fidelity were evaluated for four commercial systems. Our experimental control included CsCl-purified template DNA, Takara LA Taq polymerase, and primers and amplification protocols of Song et al. Optimization results show encouraging advances for successful application of this tool for cloning resistance genes in Solanum species, as demonstrated by our current RB allelic mining efforts. Moreover, our research increases the likelihood that LR-PCR can be applied to diverse plant species for allelic mining, isolation of functional genes, and studies on allele evolution.