Rye chromosome-specific polymerase chain reaction products developed by primers designed from theEcoO109I recognition site

Abstract

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.