Sequence divergence of rice microsatellites in Oryza and other plant species

To determine the basis of genetic variation at microsatellite loci, eleven primer pairs, developed to amplify microsatellite markers in rice, were evaluated for their ability to amplify a PCR product and for both electromorphic and sequence-based polymorphism of the resulting products in 12 plant samples, including representatives from six different species within the genus Oryza and one genotype each from Zea (maize), Triticum (wheat) and Arabidopsis. PCR amplification was reliable in the four O. sativa samples as well as in the closely related Oryza relatives with AA genomes, while only 73% (8/11) of primers amplified in the BB/CC and CC genomes of Oryza, and 27% (3/11) amplified in the other genera. Three out of seven DNA fragments that were amplified from all genera were determined to be orthologous to their rice counterparts. A total of 115 amplicons were detected using polyacrylamide gel electrophoresis and these clustered into 74 distinct electromorphs. Sequencing of 108 amplicons revealed size homoplasy, exposing 13 new sequence-based variants. Allelic diversity within a species was predominantly due to changes in the number of repeats in the microsatellite region, but the frequency of insertions/deletions (indels) and base substitutions increased as the genetic distance between samples increased. This study suggests that electromorph size polymorphism is an adequate measure of genetic difference in studies involving closely-related individuals, but that when phylogenetic or evolutionary inferences are being made over longer time scales, evaluation of SSR variation at the sequence level is essential.