Multiplex-ready markers: A technique for high-throughput, low-cost genotyping on a semi-automated DNA fragment analyser
2005
Hayden, M. | Nguyen, T. | Chalmers, K. | Australian Barley Technical Symposium (12th : 2005 : Hobart, Tasmania)
Molecular markers are widely used in agriculture, and have increased the speed and efficiency of crop improvement through marker assisted breeding. Central to the success of marker assisted breeding is the availability of faster and cheaper marker screening techniques. These techniques have become increasingly important as breeding methodologies attempt to manage and manipulate entire genomes by simultaneously using large numbers of markers to select and combine the most favorable regions of chromosomes. Here, we describe a novel multiplex PCR assay for low-cost, high-throughput marker genotyping on an automated DNA fragment analyser, such as the ABI3730. This assay allows published markers (e.g. SSRs and allele-specific STSs) to be coamplified in a single-step, closed-tube reaction under standardized conditions that require no optimization, irrespective of the optimal annealing temperature of each marker. The assay also provides several technological advantages that facilitate automated DNA fragment analysis including: (a) an ability to label each marker with a fluorescent dye of choice during multiplex PCR amplification, and (b) relatively uniform yield of PCR product for each marker, permitting the use of standardized protocols to prepare PCR fragments for DNA fragment analysis. The multiplex PCR technology is currently deployed in several Australian cereal breeding and research programs.
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