A total of 57.8 Mb of publicly available rice (Oryza sativa L.) DNA sequence was searched to determine the frequency and distribution of different simple sequence repeats (SSRs) in the genome. SSR loci were categorized into two groups based on the length of the repeat motif. Class I, or hypervariable markers, consisted of SSRs Ն20 bp, and Class II, or potentially variable markers, consisted of SSRs Ն12 bp <20 bp. The occurrence of Class I SSRs in end-sequences of EcoRI-and HindIII-digested BAC clones was one SSR per 40 Kb, whereas in continuous genomic sequence (represented by 27 fully sequenced BAC and PAC clones), the frequency was one SSR every 16 kb. Class II SSRs were estimated to occur every 3.7 kb in BAC ends and every 1.9 kb in fully sequenced BAC and PAC clones. GC-rich trinucleotide repeats (TNRs) were most abundant in protein-coding portions of ESTs and in fully sequenced BACs and PACs, whereas AT-rich TNRs showed no such preference, and di-and tetranucleotide repeats were most frequently found in noncoding, intergenic regions of the rice genome. Microsatellites with poly(AT)n repeats represented the most abundant and polymorphic class of SSRs but were frequently associated with the Micropon family of miniature inverted-repeat transposable elements (MITEs) and were difficult to amplify. A set of 200 Class I SSR markers was developed and integrated into the existing microsatellite map of rice, providing immediate links between the genetic, physical, and sequence-based maps. This contribution brings the number of microsatellite markers that have been rigorously evaluated for amplification, map position, and allelic diversity in Oryza spp. to a total of 500.[Clone sequences for 199 markers (RM1-RM88, RM200-RM345) developed in this lab are available as GenBank accessions AF343840-AF343869 and AF344003-AF344169.]Microsatellites are tandemly arranged repeats of short DNA motifs (1-6 bp in length) that frequently exhibit variation in the number of repeats at a locus. Because of their abundance and inherent potential for variation, these simple sequence repeats (SSRs) have become a valuable source of genetic markers. Previous studies in rice have contributed to the development of several hundred microsatellite markers and a genetic map consisting of 320

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