Single pollen typing combined with laser-mediated manipulation

Sachihiro Matsunaga, Karin Schutze, Iain Donnison, Sarah Grant, Tsuneyoshi Kuroiwa, Shigeyuki Kawano

Research output: Contribution to journalArticlepeer-review

39 Citations (SciVal)

Abstract

We combined single pollen typing with laser-mediated manipulation. After drilling 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 then amplified with improved primer-extension-preamplification PCR (I-PEP PCR). Nested PCR with sequence tagged site (STS)-specific primers was used to analyze several loci in the haploid genome. The single copy gene MROS1 was detected in most of the single pollen grains analyzed. Bgl10, which is localized on the Y chromosome, was detected in approximately half of the pollen grains. MROS3 is reported to be localized on the X chromosome. Using inverse PCR, we isolated two genomic clones of MROS3: MROS3A and MROS3B. The single pollen analysis using nested PCR showed that MROS3A and MROS3B are derived from different loci that are not located on the X chromosome. Single pollen typing not only reveals sex chromosome-linkage within the haploid genome, but can also discriminate between alleles and different loci. This method should also be useful for measuring recombination frequencies without genetic crossover analysis.
Original languageEnglish
Pages (from-to)371-378
Number of pages8
JournalPlant Journal
Volume20
Issue number3
DOIs
Publication statusPublished - Nov 1999

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