Frequency, distribution and sequence divergence of rice microsatellites

Microsatellites, or simple sequence repeats (SSR), are tandemly repeated DNA sequences consisting of only a few base pairs (1--6) in length. They are abundant and randomly dispersed throughout the genomes of most eukaryotic species. The DNA sequences flanking SSRs can be used to design primers for PCR (polymerase chain reaction) amplification of microsatellites. Simple sequence length polymorphisms (SSLP) can be readily detected as size differences of PCR amplified products using different DNA samples. SSLPs are valuable as genetic markers because they detect high levels of allelic diversity, are co-dominant, and are easily and economically assayed by PCR. Over the last 10 years, microsatellite markers have been developed in many plant and animal species but when this study was initiated, only 10 microsatellite had been reported in rice (Wu and Tanksley, 1993).;Over the course of this thesis research, the relative frequency of di-, tri, and tetra-nucleotide microsatellite motifs in rice was determined, a map consisting of 121 microsatellite markers was developed, and variation was examined at the sequence level to understand the nature of simple sequence length polymorphism. Based on screening of a 15 kb insert genomic library using 13 different SSR motifs, poly (GA)n appeared to be the most abundant in the rice genome and formed the foundation of the mapping effort. The relative frequency of the other repeats was observed to decrease with increasing size of the motif, though some motifs were more abundant than others in the same size class.;Clones for mapping were isolated from a short-insert, Tsp 509-digested genomic library. A total of 121 microsatellite markers were developed and integrated into an existing RFLP framework map. Mapping was accomplished using four rice populations. There was no evidence of clustering of microsatellite markers near centromeres or telomeres, and the map provided genome-wide coverage of the 12 chromosomes with an average distance of 1 SSLP (simple sequence repeat polymorphism) per 16--20 cM.;Following map development, sequence variation was investigated in the SSR motif as well as in the unique sequences flanking the SSRs. The study focused on 11 microsatellite loci using 12 plant genotypes. These included nine genotypes from within the genus, Oryza, and three genotypes from other genera, namely Zea (maize), Triticum (wheat) and Arabidopsis. At all 11 loci, allelic diversity was largely due to changes in the number of repeats in the microsatellite regions. However, additional mutational events involving insertions/deletions (indels) and base substitutions also occurred frequently in the flanking regions. Alleles of the same mw with different underlying sequences (size homoplasy) were observed among and within species.