Design and Application of Microsatellite Marker Panels for Semiautomated Genotyping of Rice (Oryza sativa L.) J. R. Coburn, S. V. Temnykh, E. M. Paul, and S. R. McCouch* ABSTRACT al. (1989). This method was then adapted and improved upon for microsatellite analysis (Edwards et al., 1991; The objective of this study was to develop a systematic and flexible Ziegle et al., 1992). Semiautomated methods of SSR method for assembling multiplex simple sequence repeat (SSR) marker panels for high throughput genome analysis in rice, Oryza genotyping are gradually replacing manual systems in sativa, and to test these panels on a set of cultivated rice germplasm. plant breeding and genetics research. These methods To do this, 159 microsatellite markers were fluorescently labeled and facilitate the efficient application of microsatellite mark- assembled into 21 multiplex panels for semiautomated genotyping, ers for high-throughput mapping (Rhodes et al., 1998; providing genome-wide coverage of the 12 rice chromosomes. Panels Ponce et al., 1999), pedigree analysis (Lexer et al., 1999), are comprised of an average of eight markers each, occurring at fingerprinting of accessions (Carrano et al., 1989), and approximately 11-centimorgan (cM) intervals throughout the genome. assaying genetic diversity (Diwan and Cregan, 1997; On a standard set of 13 genetically diverse cultivars of Oryza, these Macaulay et al., 2001). The technology can also improve markers detected an average of five alleles per locus and had a mean the efficiency of managing a germplasm collection, help polymorphism information content (P.I.C. value) of 0.67. Polymerase deliver purity-proven seed stocks to growers, and pro- chain reactions (PCR) were optimized on a per marker basis to gener- ate a uniform amount of PCR product and each primer pair was vide the basis of intellectual property protection (Mitch- assessed in replicated trials for reliability of allele size estimates. T4 ell et al., 1997). DNA polymerase was used to treat PCR products where the standard In rice, Blair et al. (2002) recently reported the use deviation of allele molecular weight was greater than 0.5 base pairs of 27 fluorescent labeled SSR markers organized into (bp). This treatment minimized the variance so that, in the multiplex four panels for diversity analysis of Oryza species. How- set reported here, the average std. dev./marker was 0.24 bp, allowing ever, to date no comprehensive, semiautomated SSR accurate discrimination of alleles that differed by a single nucleotide. genotyping system providing whole genome coverage The resulting data on allele sizes were then entered into GeneFlow has been reported for Oryza, despite the fact that ap- analysis software for the evaluation of polymorphism patterns among proximately 500 SSR markers are now publicly available diverse rice cultivars. The use of an automated software tool for for rice (Temnykh et al., 2001; http://www.gramene.org; designing multiplex panels on the basis of both highly polymorphic and more conservative SSR markers resulted in the development of verified June 12, 2002). a highly informative semiautomated genotyping system for applica- The purpose of this project was to develop and apply tions in rice genetics and breeding. multiplex panels of fluorescently labeled microsatellite markers for semiautomated genotyping of O. sativa at the whole genome level. Combinations of primer pairs T he use of fluorescently labeled microsatellite mark- were assembled to accommodate the analysis of geneti- ers for genotyping on automated sequencers offers cally diverse cultivars, providing a robust and flexible many advantages over analysis using traditional autora- approach for detection of intra- and interspecific vari- diographic or silver-stained detection techniques. One ability. The design and application of the multiplex pan- such advantage is the large increase in throughput made els was greatly facilitated by the use of the GeneFlow possible by the multiplexing of many PCR products into computer program (http://www.geneflowinc.com/; veri- a single lane. A second benefit is the significant increase fied June 12, 2002). The software allowed us the assem- in accuracy of allele sizing achieved by the use of an bly of panels in a semiautomated manner and facilitated internal size standard in each lane and the availability the analysis of the data on SSR polymorphism among of automated allele-calling algorithms. Overall, the au- accessions representing a wide spectrum of cultivated tomation increases the speed and accuracy of data col- rice germplasm. lection and processing. The high sensitivity of detection also reduces the necessary volume (and therefore the MATERIALS AND METHODS cost) of the PCR reaction and allows detection of loci Plant Material and DNA Extraction that are difficult to amplify. Use of fluorescence-based semiautomated analysis of Marker panels were tested on a standard set of 13 rice restriction fragments was first reported by Carrano et cultivars representing O. sativa spp. indica (IR36, IR64, Kasa- lath, N22, Teqing, Zhai-Ye-Qing-8, BS125), O. sativa spp. japonica (Azucena, Gihobyeo, Jing-Xi 17, Lemont, Nippon- J.R. Coburn and S.V. Temnykh, Dep. of Plant Breeding, 252 Emerson Hall, Cornell Univ., Ithaca, NY 14853-1901; E.M. Paul, GeneFlow bare), and an intermediate Korean (Tongil ) cultivar, Milyang Inc., 503 Mt. Vernon Ave., Alexandria, VA 22301; Susan R. McCouch, 23. The set has been previously used to evaluate the polymor- Dep. of Plant Breeding, 240 Emerson Hall, Cornell Univ., Ithaca, NY 14853-1901. Joint contribution of Cornell University funded by a grant Abbreviations: bp, base pair; cM, centimorgan; RCF, relative centrifu- from the Rockefeller Foundation (RF99001#726) and an unrestricted gal force, P.C.A., principle component analysis; P.I.C., polymorphism gift from RiceTec Inc. Received 25 June 2001. *Corresponding author information content; PCR, polymerase chain reaction; RFLP, restric- (SRM4@cornell.edu). tion fragment length polymorphism; RM, rice microsatellite; SSR, simple sequence repeat; WTR, well-to-read. Published in Crop Sci. 42:2092–2099 (2002). 2092 Published November, 2002