Single pollen typing combined with laser-mediated manipulation

We combined single pollen typing with laser-mediated manipulation. After dr illing a hole in the wall of a pollen grain from a dioecious plant (Silene latifolia) with a UV-laser microbeam, the single pollen grain was recovered directly in the cap of a PCR tube, using a non-contact method called laser pressure catapulting. The entire genome of the single pollen grain was the n amplified with improved primer-extension-preamplification PCR (I-PEP PCR) . Nested PCR with sequence tagged site (STS)-specific primers was used to a nalyze several loci in the haploid genome. The single copy gene MROS1 was d etected in most of the single pollen grains analyzed. Bgl10, which is local ized on the Y chromosome, was detected in approximately half of the pollen grains. MROS3 is reported to be localized on the X chromosome. Using invers e PCR, we isolated two genomic clones of MROS3: MROS3A and MROS3B. The sing le pollen analysis using nested PCR showed that MROS3A and MROS3B are deriv ed from different loci that are not located on the X chromosome. Single pol len typing not only reveals sex chromosome-linkage within the haploid genom e, but can also discriminate between alleles and different loci. This metho d should also be useful for measuring recombination frequencies without gen etic crossover analysis.