|Chen, Ping-Hua - FUJIAN AG & FORESTRY UNIV|
|Chen, Rukai - FUJIAN AG & FORESTRY UNIV|
Submitted to: Annual International Plant & Animal Genome Conference
Publication Type: Abstract Only
Publication Acceptance Date: October 1, 2007
Publication Date: January 11, 2008
Citation: Pan, Y.-B., Chen, P.-H., Chen, R.-K. 2008. A high-throughput procedure for single pollen grain collection and PCR. In: Final Abstracts Guide of the Plant and Animal Genome XVI. International Plant & Animal Genome XVI Conference, January 12-16, 2007, San Diego, California. p. 138. Technical Abstract: Pollen grains are haploid male gametophytes that share identical genetic contents of the female gametophytes; therefore, one can predict the potential genetic makeup of progeny by genotyping single pollen DNA. Amplification of DNA from single pollen grain using PCR has been accomplished in several species, however the amplification was limited to a small number of pollen grains due to difficulties in pollen isolation and lysis. The inability to conduct PCR on a large number of pollen grains has limited its application in genetic analysis and mapping studies. We developed a procedure for collecting and detecting genetic variation in a large number of individual pollen grains by PCR. The procedure involves the collection of individual pollen grains using a pair of DUMONT forceps (No. 11254-20) (Fine Science Tools USA, Inc., Foster City, CA, USA) under a dissecting microscope and the lysis of the pollen grains in a heated alkali/detergent solution followed by neutralization with a TE buffer. The resulting pollen lysates reproducibly yielded PCR products with 5S rRNA-ITS, RAPD, and SSR primers. Using this procedure, one person could collect and process up to 288 single pollen grain PCR reactions per day. The method worked well on pollen grains collected from sugarcane, corn, Miscanthus spp., snap bean, sorghum, and tomato. The ability to collect and conduct PCR on individual pollen grains on a large scale offers a new approach to genetic analyses and mapping studies in an easily controllable environment with a considerable cost reduction. The method will also benefit significantly to genetic studies in aneupolyploid plant species that are difficult subjects for classical genetic research.