title: Rye chromosome-specific polymerase chain reaction products developed by primers designed from the EcoO109I 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.
authors.affiliation: Molecular Genetics Laboratory, Faculty of Agriculture, Tottori University, 101, Minami 4-chome, Koyama-cho, Tottori, Tottori 680-8553, Japan. tomita@muses.tottori-u.ac.jp
authors.forename: Motonori
authors.initials: M
authors.lastname: Tomita
authors.forename: Akiyoshi
authors.initials: A
authors.lastname: Seno
mesh_terms: D054458:Amplified Fragment Length Polymorphism Analysis; D001483:Base Sequence; D001665:Binding Sites; D032461:Chromosomes, Plant; D017931:DNA Primers / Q000737:chemistry; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012343:RNA, Transfer / Q000737:chemistry; D012091:Repetitive Sequences, Nucleic Acid; D012434:Secale / Q000235:genetics*; D016324:Sequence Tagged Sites; D014908:Triticum / Q000235:genetics
chemical_list: D017931:DNA Primers; D012343:RNA, Transfer; D015252:Deoxyribonucleases, Type II Site-Specific; C045450:RGGNCCY-specific type II deoxyribonucleases