Rye chromosome-specific polymerase chain reaction products developed by primers designed from the EcoO109I recognition site.Genome. 2012 May; 55(5):370-82.G
From our analysis of repeat sequences in the rye genome, the presence of multiple restriction sites of EcoO109I (5'-PuGGNCCPy-3') across the genome has been predicted. By first using primers designed to contain EcoO109I sites in polymerase chain reaction (PCR), polymorphic DNA markers were effectively obtained. A total of 43 types of 10-mer primers containing EcoO109I sites were applied for PCR by using genomic DNA of Secale cereale self-fertile line IR27 and Triticum aestivum 'Chinese Spring' (CS) as the template. Twenty two primers detected polymorphisms between wheat and rye, and they were applied for PCR using a series of CS wheat--'Imperial' rye chromosome addition lines as templates. Nine chromosome-specific amplification fragments identified on five chromosomes were collected from gels and hybridized with nylon membrane-transferred PCR products from the wheat-rye chromosome addition lines. The gel blot was only observed between the collected fragments; therefore, these fragments were confirmed to be chromosome-specific. These fragments were sequenced and converted to sequence-tagged site (STS) primers. We therefore introduce a new method for building chromosome-specific DNA markers: (i) multiple polymorphic fragments can be obtained from EcoO109I primers and (ii) the addition of three nucleotides to the EcoO109I site restricts the amplification region to generate chromosome-specific fragments.