In three-line system, cytoplasmic male sterile (CMS) lines often were contaminated with cognate iso-nuclear maintainer lines during seeds multiplication processes. Therefore fingerprinting of breeding lines and identification of line-specific markers are prerequisite in genetic purity test. Six CMS lines including Neda-A, Nemat-A, Dasht-A, Amol 3-A, Champa-A, IR58025A and their iso-nuclear maintainers were used in this study. Twenty-five random amplified polymorphic DNA (RAPD) primers used for screening CMS lines and maintainer lines. The result indicated that the iso-gene lines had similar band patterns in most marker loci due to similar genetic background that present between CMS lines and co-maintainer lines. However two specific bands, 950 bp and 850 bp were produced by OPH20 and OPH01 primers that could uniquely recognize the CMS lines from their cognate maintainer lines, respectively. The genetic nature of OPH20-950 band was determinated by isolation of fragment from gel, clone to Escherichia coli bacteria via T/A cloning system, and was sequenced. A BLAST search of OPH20-950 sequence with GenBank indicated 95% homology to a rice mitochondrial DNA. These line-specific fragments could be used as a specific feature (scare marker) for characterization and genetic purity test of WA CMS lines.

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