Design and application of microsatellite marker panels for semiautomated genotyping of rice (Oryza sativa L.)

The objective of this study was to develop a systematic and flexible method for assembling multiplex simple sequence repeat (SSR) marker panels for high throughput genome analysis in rice, Oryza sativa, and to test these panels on a set of cultivated rice germplasm. To do this, 159 microsatellite markers were fluorescently labeled and assembled into 21 multiplex panels for semiautomated genotyping, providing genome-wide coverage of the 12 rice chromosomes. Panels are comprised of an average of eight markers each, occurring at approximately 11-centimorgan (cM) intervals throughout the genome. On a standard set of 13 genetically diverse cultivars of Oryza, these markers detected an average of five alleles per locus and had a mean polymorphism information content (P.I.C. value) of 0.67. Polymerase chain reactions (PCR) were optimized on a per marker basis to generate a uniform amount of PCR product and each primer pair was assessed in replicated trials for reliability of allele size estimates. T4 DNA polymerase was used to treat PCR products where the standard deviation of allele molecular weight was greater than 0.5 base pairs (bp). This treatment minimized the variance so that, in the multiplex set reported here, the average std. dev./marker was 0.24 bp, allowing accurate discrimination of alleles that differed by a single nucleotide. The resulting data on allele sizes were then entered into GeneFlow analysis software for the evaluation of polymorphism patterns among diverse rice cultivars. The use of an automated software tool for designing multiplex panels on the basis of both highly polymorphic and more conservative SSR markers resulted in the development of a highly informative semiautomated genotyping system for applications in rice genetics and breeding.