Cloning of random amplified polymorphic DNA molecular markers useful to introduce alien genes for wheat breeding.

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Oligonucleotide primers were designed from the recognition sites of the restriction enzyme EcoO1091 and enabled in polymerase chain reaction (PCR) to amplify chromosome-specific DNA sequences of rye, Secale cereale, being an useful genetic stock for wheat breeding.TOMITA et al. (1993) found that EcoO1091 recognition site were frequently distributed in the rye genome. The objective of the present study is to explore the use of the EcoO1091 recognition site as PCR primers to amplify alien (rye) sequences in whert genetic backgrounds. Genomic DNAs were isolated from yong leaves of a rye inbred line IR27, a wheat cv. Chiness Spring (CS) and CS-rye cv. Imperial chromosome addition lines. Forty-five kinds of 10 mer primers were synthesized by a Gene Assembler (Pharmacia). PCR were conducted in a total volume of 50 mu l with 20ng genomic DNA,0.2 mu M of 10 mer primer, 100 mu M of each deoxyribonucleotide, 1 unit of Taq polymerase and 1*Taq PCR buffer. The PCR reactions were performed in a The … More rmal Cycler PC-700 (ASTEC) for 45 cycles of 1 min at 97ﾟC for denaturation, 1 min at 36ﾟC for annealing and 2 min at 72ﾟC for extension. Ten mu l of the PCR products were separated on 1.5% agarose gels in 1*TAE buffer. Ten kinds of fragments whose size is specific to rye chromosome 1R,3R,4R,5R and 7R were found in the PCR products from seven kinds of Wheat-Rye chromosome addition lines. These rye chromosome-specific amplification were recovered from agarose gel and used as probes for Southern Hybridization to be PCR products from Wheat-Rye chromosome addition lines. Recovered DNA fragments hybridized to each fragment itself and did not exhibit hybridization signal in the other PCR products especially from wheat, indicating that rye chromosome-specific amplifications were unique to rye chromosomes. These rye chromosome-specific sequences were cloned with pMOS Blue and sequenced using by an A.L.F DNA sequencer (Pharmacia). The 3R and 5R chromosome-specific amplifications showed tRNA-like structure which is often discovered in animal dispersed repeated DNA sequences "retroposon" and smear hybridization patterns to all kinds of Wheat-Rye chromosome addition lines. This finding indicated that rye chromosome-specific amplifications were produced by internal variations of the EcoO1091 recognition sites in dispersed repeated sequences.EcoO1091 primers allowed identification of rye chromosomes in wheat-rye addition lines and also demonstrated significant polymorphism among diverse sources of rye. This approach, PCR using DNA primers designed from the EcoO1091 site, was shown to be an useful method for the identification of alien (rye) DNA in wheat genetic backgrounds and wheat lines carrying rye chromosomes. Less