Inter-simple sequence repeat (ISSR) amplification for analysis of microsatellite motif frequency and fingerprinting in rice (Oryza sativa L.)

Inter-simple sequence repeat (ISSR) amplification was used to analyze micro satellite motif frequency in the rice genome and to evaluate genetic divers ity among rice cultivars. A total of 32 primers, containing different simpl e sequence repeat (SSR) motifs, were tested for amplification on a panel of 59 varieties. representative of the diversity of cultivated rice (Oryza sa tiva L.). The ISSR analysis provided insights into the organization, freque ncy and levels of polymorphism of different simple sequence repeats in rice . The more common dinucleotide motifs were more amenable to ISSR analysis t han the more infrequent tri-, tetra- and penta-nucleotide motifs. The ISSR results suggested that within the dinucleotide class, the poly(GA) motif wa s more common than the poly(GT) motif and that the frequency and clustering of specific tri- and tetra-nucleotide simple sequence repeats was variable and motif-specific. Furthermore, trinucleotide ISSR markers were found to be less polymorphic than either dinucleotide or certain tetranucleotide ISS R markers, suggesting which motifs would be better targets for microsatelli te marker development. The ISSR amplification pattern was used to group the rice genotypes by cluster analysis. These results were compared to surveys of the same varieties for amplified fragment length polymorphism (AFLP), r estriction fragment length polymorphism (RFLP) and isozyme markers. The ISS R fingerprint could be used to differentiate the genotypes belonging to eit her Japonica or Indica subspecies of cultivated rice and to dissect finer l evels of diversity within each subspecies. A higher percentage of polymorph ic bands was produced with the ISSR technique than the AFLP method, based o n a similar PCR reaction. Therefore, ISSR amplification proved to be a valu able method for determining genetic variability among rice varieties and fo r rapidly identifying cultivars. This efficient genetic fingerprinting tech nique would be useful for characterizing the large numbers of rice accessio ns held in national and international germplasm centers.