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High output genetic mapping of polyploids using PCR-generated markers
Authors:Bruno W S Sobral  Rhonda J Honeycutt
Institution:(1) California Institute of Biological Research, 11099 North Torrey Pines Road, Suite 300, 92037 La Jolla, CA, USA
Abstract:Summary The polymerase chain reaction (PCR) with arbitrarily selected primers has been established as an efficient method to generate fingerprints that are useful in genetic mapping and genomic fingerprinting. To further increase the productivity of mapping and fingerprinting efforts, we have altered existing protocols to include the use of the Stoffel fragment, which is derived from genetically engineered Taq polymerase. We also optimized the thermal profile of the reaction to increase the number of useful primers. In mapping of the genome of Saccharum spontaneum lsquoSES 208rsquo, a polyploid wild relative of sugarcane, these modifications allowed for an increase of 30% in the number of loci screened per primer, and an 80% increase in the number of polymorphisms per primer. Furthermore, the enzyme cost per reaction was decreased approximately 1.6-fold. Finally, there was an increase from about 70% to about 97% in the number of primers that were useful (i.e., gave a reproducible fingerprint) using our protocol. We have placed some of these markers into linkage groups.
Keywords:AP-PCR  RAPD  Sugarcane  Saccharum  Stoffel fragment  Genetic mapping  MapMaker  Molecular markers
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