One of main problems in three-line hybrid seed production of tobacco (Nicotinia tabacum) is differentiation between fertile maintainer lines and CMS counterparts that is indispensable for seed purity tests. To establish a reliable marker system namely amplified length polymorphism (ALP) for differentiating between normal fertile tobacco lines and CMS counterparts, two CMSspecific primers (designates as Ntmto1F and ntmto1R) were designed based on the alignment of the mitochondrial DNA sequence of two Gene Bank accessions (AF056245 and AF121902). Preliminary PCR tests by this primer pair on the DNA of three fertile lines and CMS counterparts from three well-known tobacco ecotypes (Burley, Basma and Virginia) and subsequent silver staining after vertical electrophoresis indicated the usefulness of the developed ALP marker system for distinguishing between two fertile and CMS counterparts. Normal fertile lines amplified a shorter fragment than the respective CMS counterparts. To validate the marker system, DNA from further fertile and CMS lines of three ecotypes were subjected to PCR amplification by the primer pair. Results showed that this marker system could discriminate two fertile and CMS groups with 100% carefulness. This marker system is highly reliable and costeffective and can be utilized by plant breeders and hybrid seed producers.
