Submitted to: Journal of Integrative Plant Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/5/2007
Publication Date: 3/18/2008
Citation: Chen, P.-H., Pan, Y.-B., Chen, R.-K. 2008. High-throughput procedure for single pollen grain collection and polymerase chain reaction in plants. Journal of Integrative Plant Biology. 50:375-383. Interpretive Summary: In general, sugarcane crosses produce three classes of offspring. Class I offspring are true hybrids that produce DNA fingerprints found in both parents; Class II offspring produce DNA fingerprints not found in either parent as a result of pollination from pollen of an unknown source; and Class III offspring produce DNA fingerprints only found in the maternal parent. Breeders are not certain if Class III offspring are self-pollinated progeny of the female parent or true hybrids from the designated pollen source that do not inherit the DNA fingerprints. One solution to this problem is to study how the DNA fingerprints pass through from the vegetative clones into the reproductive cells, i.e., female eggs or male pollen grains, through polymerase chain reaction (PCR)-based DNA fingerprinting analysis. While access to the eggs is destructive to the sugarcane plant, pollen grains are abundant and easily accessible. However, the numbers of pollen grains that have been used in such studies are limited due to difficulties in pollen isolation and lysis. Using a pair of special forceps and a 63-fold magnification, we are able to collect an average of 60 pollen grains per hour. We also are able to obtain the DNA material from the single pollen grains by a high-throughput denaturation and neutralization process. Under optimized PCR conditions, we can produce DNA fingerprinting information from up to 288 single pollen grains per day. We have demonstrated that the high-throughput single pollen grain fingerprinting procedure works equally well on corn, Miscanthus spp., snap bean, sorghum, and tomato with five different DNA markers. The ability to collect and conduct PCR on individual pollen grains on a large scale offers a new approach to genetic analyses in sugarcane and other multiplexed species that are difficult subjects for classical genetic research.
Technical Abstract: Single pollen grain PCR has succeeded in several species, however only a limited number (up to 60) pollen grains were involved due to difficulties in pollen isolation and lysis. This has limited its application in genetic analysis and mapping studies in plants. A high-throughput procedure for detecting genetic variation in individual pollen grains of sugarcane by polymerase chain reaction (PCR) is reported. The procedure involved the collection of individual pollen grains under magnification by a pair of special forceps and the lysis of the pollen grains in an alkali/detergent solution followed by neutralization with TE buffer. The resulting template solutions yielded PCR reactions involving the 5S rRNA intergenic transcribed spacer, two RAPD, and two microsatellite markers. PCR parameters were optimized without the need of pre-amplification. Using this procedure, one person with experience could process up to 288 single pollen grain PCR reactions per day. The method also worked well on five other species, namely, corn, Miscanthus spp., snap bean, sorghum, and tomato. The ability to collect pollen and conduct PCR on individual pollen grains on a large scale offers a new approach to genetic analyses in sugarcane and other species that are difficult subjects for classical genetic research.