Application of multiplex PCR and fluorescence-based, semi-automated allele sizing technology for genotyping plant genetic resources

For future progress, effective conservation of plant genetic resources will require the integration of technologies and protocols that provide for the acquisition of large quantities of genetic information for improved gene and genotype identification. As a contribution to this integration, simple sequence repeat (SSR) marker analysis in Brassica spp. was evaluated as a prototype system for establishing the potential of multiplex polymerase chain reactions (PCRs) coupled with fluorescence-based DNA detection and semi-automated sizing technology for mass genotyping of plant genetic resources. Consistent results were achieved for multiplex PCRs (simultaneous amplification of several genetic markers in a single reaction) with 11 fluorescently labeled primer pairs. Comparison of computer-derived estimates of fragment sizes between gels demonstrated that automated sizing was accurate enough to achieve reliable and repeatable plant genotypes. To establish a genetic basis for putative SSR loci previously identified in Brassica spp., segregation data were analyzed. Three polymorphic SSRs in Brassica napus L. (canola and rapeseed) exhibited simple Mendelian inheritance and were not linked. One of these polymorphic loci was duplicated, i.e., a single copy marked each of the ''aa'' and ''cc'' genomes originating from progenitor species B. rapa and B. oleracea, respectively. Technologies that provide for high genetic resolution and throughput at reasonable costs will find ready application in plant genetic resources conservation. In addition, these same tools and marker systems can be applied to questions of pedigree analysis and intellectual property rights.