Amplification of RAPD markers from single pollen grains of diploid (==24) potato

Pollen grains from a diploid clone 9507-04 were used for RAPD analysis. Single pollen grains were isolated from pollen in liquid suspension, or from dry pollen spread on a glass slide, and collected in individual PCR tubes. Dry pollen isolation was found advantageous due to the convenience of handling and reduction in DNA cross-contamination. Primers UBC291 and UBC504 were selected for the RAPD analysis of both the pollen samples and the pollen donor. clone 9507-04 produced three RAPD markers UBC291-1.4, UBC291-0.8 and UBC291-0.4, under the selected PCR conditions. Amplification of three additional parental markers UBC504-1.17, UBC504-0.96 and UBC504-0.78 was also tested in a selected pollen sample. RAPD products were discernible from single pollen grains without requiring any DNA extraction procedures. The three UBC291 amplified RAPD markers of the parent segregated in the selected sample of 60 pollen grains. Potential applications of the RAPD analysis of single pollen grains are described.