Abstract The growing number of rice microsatellite markers warrants a comprehensive comparison of allelic variability between the markers developed using differ- ent methods, with various sequence repeat motifs, and from coding and non-coding portions of the genome. We have performed such a comparison over a set of 323 microsatellite markers; 194 were derived from genomic library screening and 129 were derived from the analysis of rice-expressed sequence tags (ESTs) available in public DNA databases. We have evaluated the frequency of polymorphism between parental pairs of six inter- subspecific crosses and one inter-specific cross widely used for mapping in rice. Microsatellites derived from genomic libraries detected a higher level of polymor- phism than those derived from ESTs contained in the GenBank database (83.8% versus 54.0%). Similarly, the other measures of genetic variability [the number of al- leles per locus, polymorphism information content (PIC), and allele size ranges] were all higher in genomic libra- ry-derived microsatellites than in their EST-database counterparts. The highest overall degree of genetic di- versity was seen in GA-containing microsatellites of ge- nomic library origin, while the most conserved markers contained CCG- or CAG-trinucleotide motifs and were developed from GenBank sequences. Preferential loca- tion of specific motifs in coding versus non-coding re- gions of known genes was related to observed levels of microsatellite diversity. A strong positive correlation was observed between the maximum length of a microsatel- lite motif and the standard deviation of the molecular- weight of amplified fragments. The reliability of molecu- lar weight standard deviation (SDmw) as an indicator of genetic variability of microsatellite loci is discussed. Key words Allelic diversity · Simple sequence repeat (SSR) · Microsatellite marker · Rice (Oryza sativa L.) Introduction Microsatellites, also known as simple sequence repeats (SSRs), are tandem arrays of short nucleotide repeats from 1 to 5 bases per unit. Simple sequence length polymor- phisms (SSLPs) are based on the difference in the number of the DNA repeat units at a given locus and provide a valuable source of genetic markers. Microsatellites have been extensively exploited for genome mapping and for a wide range of population and evolutionary studies in hu- man (Bowcock et al. 1994; Dib et al. 1996), mouse (Dietrich et al. 1996), Drosophila (Goldstein and Clark 1995; Schlötterer et al. 1997; Schug et al. 1998), Arab- idopsis (Innan et al. 1997), rice (Yang et el. 1994), and other animal and plant species (Jarne and Lagoda 1996; Powell et al. 1996). In addition to its clear utility for prac- tical applications such as genetic mapping and fingerprint- ing, information about the distribution and variability of microsatellite sequences in the genome of a given species can elucidate the genetic history of the species from the standpoint of evolution and artificial selection. Population Communicated by F. Salamini The first three authors made equal contributions to this manuscript Y.G. Cho · T. Ishii · S. Temnykh · X. Chen · L. Lipovich S.R. McCouch ( ✉ ) Department of Plant Breeding, 252 Emerson Hall, Cornell University, Ithaca, NY 14853–1901, USA W.D. Park · N. Ayres · S. Cartinhour Department of Biochemistry and Biophysics, Texas A&M University, College Station, TX 77845, USA Present addresses: Y.G. Cho, Department of Agronomy, Chungbuk National University, Chongju 361–763, Korea T. Ishii, Laboratory of Plant Breeding, Faculty of Agriculture, Kobe University, Nada-ku, Kobe 657–8501, Japan L. Lipovich, Department of Molecular Biotechnology, University of Washington, 1705 NE Pacific St., Box 357730, Seattle, WA 98195–7730, USA S. Cartinhour, USDA/ARS and Department of Plant Breeding, G16 Bradfield Hall, Cornell University, Ithaca, NY 14853–1901, USA Theor Appl Genet (2000) 100:713–722 © Springer-Verlag 2000 ORIGINAL PAPER Y.G. Cho · T. Ishii · S. Temnykh · X. Chen L. Lipovich · S.R. McCouch · W.D. Park · N. Ayres S. Cartinhour Diversity of microsatellites derived from genomic libraries and GenBank sequences in rice (Oryza sativa L.) Received: 5 May 1999 / Accepted: 16 August 1999